Peptides and related molecules that bind to tall-1

ABSTRACT

The present invention concerns therapeutic agents that modulate the activity of TALL-1. In accordance with the present invention, modulators of TALL-1 may comprise an amino acid sequence Dz 2 Lz 4  wherein z 2  is an amino acid residue and z 4  is threonyl or isoleucyl. Exemplary molecules comprise a sequence of the formulae 
                         (SEQ. ID. NO: 100)         a 1 a 2 a 3 CDa 6 La 8 a 9 a 10 Ca 12 a 13 a 14 ,                   (SEQ. ID. NO: 104)         b 1 b 2 b 3 Cb 6 Db 8 Lb 10 b 11 b 12 b 13 b 14 Cb 16 b 17 b 18                     (SEQ. ID. NO: 105)         c 1 c 2 c 3 Cc 5 Dc 7 Lc 9 c 10 c 11 c 12 c 13 c 14 Cc 16 c 17 c 18                     (SEQ. ID. NO: 106)         d 1 d 2 d 3 Cd 5 d 6 d 7 WDd 10 Ld 13 d 14 d 15 Ld 16 d 17 d 18                     (SEQ. ID. NO: 107)         e 1 e 2 e 3 Ce 5 e 6 e 7 De 9 Le 11 Ke 13 ce 15 e 16 e 17 e 18                     (SEQ. ID NO: 109)         f 1 f 2 f 3 Kf 5 Df 7 Lf 9 f 10 Qf 12 f 13 f 14                                 
wherein the substituents are as defined in the specification. The invention further comprises compositions of matter of the formula
 
       (X 1 ) a —V 1 —(X 2 ) b  
 
     wherein V1 is a vehicle that is covalently attached to one or more of the above TALL-1 modulating compositions of matter. The vehicle and the TALL-1 modulating composition of matter may be linked through the N- or C-terminus of the TALL-1 modulating portion. The preferred vehicle is an Fc domain, and the preferred Fc domain is an IgG Fc domain.

RELATED APPLICATIONS

The present application is a continuation of U.S. patent applicationSer. No. 13/938,141, filed Jul. 9, 2013, which is a divisional of U.S.patent application Ser. No. 12/788,137, filed May 26, 2010, issued asU.S. Pat. No. 8,507,426, issued Aug. 13, 2013, which is a continuationof U.S. patent application Ser. No. 11/272,521, filed Nov. 10, 2005,issued as U.S. Pat. No. 7,737,111, issued Jun. 15, 2010, which is adivisional of U.S. patent application Ser. No. 10/145,206, filed May 13,2002, issued as U.S. Pat. No. 7,259,137, issued Aug. 21, 2007, whichclaims priority to U.S. Provisional Application No. 60/290,196, filedMay 11, 2001, the disclosures of which are incorporated by referenceherein in their entirety, including drawings.

BACKGROUND OF THE INVENTION

After years of study in necrosis of tumors, tumor necrosis factors(TNFs) α and β were finally cloned in 1984. The ensuing years witnessedthe emergence of a superfamily of TNF cytokines, including fas ligand(FasL), CD27 ligand (CD27L), CD30 ligand (CD30L), CD40 ligand (CD40L),TNF-related apoptosis-inducing ligand (TRAIL, also designated AGP-1),osteoprotegerin binding protein (OPG-BP or OPG ligand), 4-1BB ligand,LIGHT, APRIL, and TALL-1. Smith et al. (1994), Cell 76: 959-962; Laceyet al. (1998), Cell 93: 165-176; Chichepotiche et al. (1997), J. Biol.Chem. 272: 32401-32410; Mauri et al. (1998), Immunity 8: 21-30; Hahne etal. (1998), J. Exp. Med. 188: 1185-90; Shu et al. (1999), J. LeukocyteBiology 65: 680-3. This family is unified by its structure, particularlyat the C-terminus. In addition, most members known to date are expressedin immune compartments, although some members are also expressed inother tissues or organs, as well. Smith et al. (1994), Cell 76: 959-62.All ligand members, with the exception of LT-α, are type IItransmembrane proteins, characterized by a conserved 150 amino acidregion within C-terminal extracellular domain. Though restricted to only20-25% identity, the conserved 150 amino acid domain folds into acharacteristic β-pleated sheet sandwich and trimerizes. This conservedregion can be proteolytically released, thus generating a solublefunctional form. Banner et al. (1993), Cell 73: 431-445.

Many members within this ligand family are expressed in lymphoidenriched tissues and play important roles in the immune systemdevelopment and modulation. Smith et al. (1994). For example, TNFα ismainly synthesized by macrophages and is an important mediator forinflammatory responses and immune defenses. Tracey & Cerami (1994), Ann.Rev. Med. 45: 491-503. Fas-L, predominantly expressed in activated Tcell, modulates TCR-mediated apoptosis of thymocytes. Nagata, S. & Suda,T. (1995) Immunology Today 16: 39-43; Castrim et al. (1996), Immunity 5:617-27. CD40L, also expressed by activated T cells, provides anessential signal for B cell survival, proliferation and immunoglobulinisotype switching. Noelle (1996), Immunity 4: 415-9.

The cognate receptors for most of the TNF ligand family members havebeen identified. These receptors share characteristic multiplecysteine-rich repeats within their extracellular domains, and do notpossess catalytic motifs within cytoplasmic regions. Smith et al.(1994). The receptors signal through direct interactions with deathdomain proteins (e.g. TRADD, FADD, and RIP) or with the TRAF proteins(e.g. TRAF2, TRAF3, TRAF5, and TRAF6), triggering divergent andoverlapping signaling pathways, e.g. apoptosis, NF-_(K)B activation, orJNK activation. Wallach et al. (1999), Annual Review of Immunology 17:331-67. These signaling events lead to cell death, proliferation,activation or differentiation. The expression profile of each receptormember varies. For example, TNFR1 is expressed on a broad spectrum oftissues and cells, whereas the cell surface receptor of OPGL is mainlyrestricted to the osteoclasts. Hsu et al. (1999) Proc. Natl. Acad. Sci.USA 96: 3540-5.

A number of research groups have recently identified TNF family ligandswith the same or substantially similar sequence. The ligand has beenvariously named neutrokine a (WO 98/18921, published May 7, 1998), 63954(WO 98/27114, published Jun. 25, 1998), TL5 (EP 869 180, published Oct.7, 1998), NTN-2 (WO 98/55620 and WO 98/55621, published Dec. 10, 1998),TNRL1-alpha (WO 9911791, published Mar. 11, 1999), kay ligand(WO99/12964, published Mar. 18, 1999), and AGP-3 (U.S. Prov. App. Nos.60/119,906, filed Feb. 12, 1999 and 60/166,271, filed Nov. 18, 1999,respectively); and TALL-1 (WO 00/68378, published Nov. 16, 2000). Eachof these references is hereby incorporated by reference. Hereinafter,the ligands reported therein are collectively referred to as TALL-1.

TALL-1 is a member of the TNF ligand superfamily that is functionallyinvolved in B cell survival and proliferation. Transgenic miceoverexpressing TALL-1 had severe B cell hyperplasia and lupus-likeautoimmune disease. Khare et al. (2000) PNAS 97(7):3370-3375). Both TACIand BCMA serve as cell surface receptors for TALL-1. Gross et al.(2000), Nature 404: 995-999; Ware (2000), J. Exp. Med. 192(11): F35-F37;Ware (2000), Nature 404: 949-950; Xia et al. (2000), J. Exp. Med.192(1):137-143; Yu et al. (2000), Nature Immunology 1(3):252-256;Marsters et al. (2000), Current Biology 10:785-788; Hatzoglou et al.(2000) J. of Immunology 165:1322-1330; Shu et al. (2000) PNAS97(16):9156-9161; Thompson et al. (2000) J. Exp. Med. 192(1):129-135;Mukhopadhyay et al. (1999) J. Biol. Chem. 274(23): 15978-81; Shu et al.(1999) J. Leukocyte Biol. 65:680-683; Gruss et al. (1995) Blood 85(12):3378-3404; Smith et al. (1994), Cell 76: 959-962; U.S. Pat. No.5,969,102, issued Oct. 19, 1999; WO 00/67034, published Nov. 9, 2000; WO00/40716, published Jul. 13, 2000; WO 99/35170, published Jul. 15, 1999.Both receptors are expressed on B cells and signal through interactionwith TRAF proteins. In addition, both TACI and BCMA also bind to anotherTNF ligand family member, APRIL. Yu et al. (2000), Nature Immunology1(3):252-256. APRIL has also been demonstrated to induce B cellproliferation.

To date, no recombinant or modified proteins employing peptidemodulators of TALL-1 have been disclosed. Recombinant and modifiedproteins are an emerging class of therapeutic agents. Usefulmodifications of protein therapeutic agents include combination with the“Fc” domain of an antibody and linkage to polymers such as polyethyleneglycol (PEG) and dextran. Such modifications are discussed in detail ina patent application entitled, “Modified Peptides as TherapeuticAgents,” publicshed WO 00/24782, which is hereby incorporated byreference in its entirety.

A much different approach to development of therapeutic agents ispeptide library screening. The interaction of a protein ligand with itsreceptor often takes place at a relatively large interface. However, asdemonstrated for human growth hormone and its receptor, only a few keyresidues at the interface contribute to most of the binding energy.Clackson et al. (1995), Science 267: 383-6. The bulk of the proteinligand merely displays the binding epitopes in the right topology orserves functions unrelated to binding. Thus, molecules of only “peptide”length (2 to 40 amino acids) can bind to the receptor protein of a givenlarge protein ligand. Such peptides may mimic the bioactivity of thelarge protein ligand (“peptide agonists”) or, through competitivebinding, inhibit the bioactivity of the large protein ligand (“peptideantagonists”).

Phage display peptide libraries have emerged as a powerful method inidentifying such peptide agonists and antagonists. See, for example,Scott et al. (1990), Science 249: 386; Devlin et al. (1990), Science249: 404; U.S. Pat. No. 5,223,409, issued Jun. 29, 1993; U.S. Pat. No.5,733,731, issued Mar. 31, 1998; U.S. Pat. No. 5,498,530, issued Mar.12, 1996; U.S. Pat. No. 5,432,018, issued Jul. 11, 1995; U.S. Pat. No.5,338,665, issued Aug. 16, 1994; U.S. Pat. No. 5,922,545, issued Jul.13, 1999; WO 96/40987, published Dec. 19, 1996; and WO 98/15833,published Apr. 16, 1998 (each of which is incorporated by reference inits entirety). In such libraries, random peptide sequences are displayedby fusion with coat proteins of filamentous phage. Typically, thedisplayed peptides are affinity-eluted against an immobilized targetprotein. The retained phages may be enriched by successive rounds ofaffinity purification and repropagation. The best binding peptides maybe sequenced to identify key residues within one or more structurallyrelated families of peptides. See, e.g., Cwirla et al. (1997), Science276: 1696-9, in which two distinct families were identified. The peptidesequences may also suggest which residues may be safely replaced byalanine scanning or by mutagenesis at the DNA level. Mutagenesislibraries may be created and screened to further optimize the sequenceof the best binders. Lowman (1997), Ann. Rev. Biophys. Biomol. Struct.26: 401-24.

Structural analysis of protein-protein interaction may also be used tosuggest peptides that mimic the binding activity of large proteinligands. In such an analysis, the crystal structure may suggest theidentity and relative orientation of critical residues of the largeprotein ligand, from which a peptide may be designed. See, e.g.,Takasaki et al. (1997), Nature Biotech. 15: 1266-70. These analyticalmethods may also be used to investigate the interaction between areceptor protein and peptides selected by phage display, which maysuggest further modification of the peptides to increase bindingaffinity.

Other methods compete with phage display in peptide research. A peptidelibrary can be fused to the carboxyl terminus of the lac repressor andexpressed in E. coli. Another E. coli-based method allows display on thecell's outer membrane by fusion with a peptidoglycan-associatedlipoprotein (PAL). Hereinafter, these and related methods arecollectively referred to as “E. coli display.” In another method,translation of random RNA is halted prior to ribosome release, resultingin a library of polypeptides with their associated RNA still attached.Hereinafter, this and related methods are collectively referred to as“ribosome display.” Other methods employ peptides linked to RNA; forexample, PROfusion technology, Phylos, Inc. See, for example, Roberts &Szostak (1997), Proc. Natl. Acad. Sci. USA, 94: 12297-303. Hereinafter,this and related methods are collectively referred to as “RNA-peptidescreening.” Chemically derived peptide libraries have been developed inwhich peptides are immobilized on stable, non-biological materials, suchas polyethylene rods or solvent-permeable resins. Another chemicallyderived peptide library uses photolithography to scan peptidesimmobilized on glass slides. Hereinafter, these and related methods arecollectively referred to as “chemical-peptide screening.”Chemical-peptide screening may be advantageous in that it allows use ofD-amino acids and other unnatural analogues, as well as non-peptideelements. Both biological and chemical methods are reviewed in Wells &Lowman (1992), Curr. Opin. Biotechnol. 3: 355-62. Conceptually, one maydiscover peptide mimetics of any protein using phage display,RNA-peptide screening, and the other methods mentioned above.

SUMMARY OF THE INVENTION

The present invention concerns therapeutic agents that modulate theactivity of TALL-1. In accordance with the present invention, modulatorsof TALL-1 may comprise an amino acid sequence Dz²Lz⁴ (SEQ ID NO: 108)wherein z² is an amino acid residue and z⁴ is threonyl or isoleucyl.Such modulators of TALL-1 comprise molecules of the following formulae:

I(a)  (SEQ. ID. NO: 100) a¹a²a³CDa⁶La⁸a⁹a¹⁰Ca¹²a¹³a¹⁴wherein:

-   -   a¹, a², a³ are each independently absent or amino acid residues;    -   a⁶ is an amino acid residue;    -   a⁹ is a basic or hydrophobic residue;    -   a⁸ is threonyl or isoleucyl;    -   a¹² is a neutral hydrophobic residue; and    -   a¹³ and a¹⁴ are each independently absent or amino acid        residues.

I(b)  (SEQ. ID. NO: 104) b¹b²b³Cb⁵b⁶Db⁸Lb¹⁰b¹¹b¹²b¹³b¹⁴Cb¹⁶b¹⁷b¹⁸wherein:

-   -   b¹ and b² are each independently absent or amino acid residues;    -   b³ is an acidic or amide residue;    -   b⁵ is an amino acid residue;    -   b⁶ is an aromatic residue;    -   b⁸ is an amino acid residue;    -   b¹⁰ is T or I;    -   b¹¹ is a basic residue;    -   b¹² and b¹³ are each independently amino acid residues;    -   b¹⁴ is a neutral hydrophobic residue; and    -   b¹⁶, b¹⁷, and b¹⁸ are each independently absent or amino acid        residues.

I(c) (SEQ. ID. NO: 105) c¹c²c³Cc⁵Dc⁷Lc⁹c¹⁰c¹¹c¹²c¹³c¹⁴Cc¹⁶c¹⁷c¹⁸wherein:

-   -   c¹, c², and c³ are each independently absent or amino acid        residues;    -   c⁵ is an amino acid residue;    -   c⁷ is an amino acid residue;    -   c⁹ is T or I;    -   c¹⁰ is a basic residue;    -   c¹¹ and c¹² are each independently amino acid residues;    -   c¹³ is a neutral hydrophobic residue;    -   c¹⁴ is an amino acid residue;    -   c¹⁶ is an amino acid residue;    -   c¹⁷ is a neutral hydrophobic residue; and    -   c¹⁸ is an amino acid residue or is absent.

I(d) (SEQ. ID. NO: 106) d¹d²d³Cd⁵d⁶d⁷WDd¹⁰Ld¹²d¹³d¹⁴Cd¹⁵d¹⁶d¹⁷wherein:

-   -   d¹, d², and d³ are each independently absent or amino acid        residues;    -   d⁵, d⁶, and d⁷ are each independently amino acid residues;    -   d¹⁰ is an amino acid residue;    -   d¹³ is T or I;    -   d¹⁴ is an amino acid residue; and    -   d¹⁶, d¹⁷, and d¹⁸ are each independently absent or amino acid        residues.

I(e) (SEQ. ID. NO: 107) e¹e²e³Ce⁵e⁶e⁷De⁹Le¹¹Ke¹³Ce¹⁵e¹⁶e¹⁷e¹⁸wherein:

-   -   e¹, e², and e³ are each independently absent or amino acid        residues;    -   e⁵, e⁶, e⁷, e⁹, and e¹³ are each independently amino acid        residues;    -   e¹¹ is T or I; and    -   e¹⁵, e¹⁶, and e¹⁷ are each independently absent or amino acid        residues.

I(f) (SEQ. ID NO: 109) f¹f²f³Kf⁵Df⁷Lf⁹f¹⁰Qf¹²f¹³f¹⁴wherein:

-   -   f¹, f², and f³ are absent or are amino acid residues (with one        of f¹, f², and f³ preferred to be C when one of f¹², f¹³, and        f¹⁴ is C);    -   f⁵ is W, Y, or F (W preferred);    -   f⁷ is an amino acid residue (L preferred);    -   f⁹ is T or I (T preferred);    -   f¹⁰ is K, R, or H (K preferred);    -   f¹² is C, a neutral hydrophobic residue, or a basic residue (W,        C, or R preferred);    -   f¹³ is C, a neutral hydrophobic residue or is absent (V        preferred); and    -   f¹⁴ is any amino acid residue or is absent;    -   provided that only one of f¹, f², and f³ may be C, and only one        of f¹², f¹³, and f¹⁴ may be C.

Compounds of formulae I(a) through I(f) above incorporate Dz²Lz⁴, aswell as SEQ ID NO: 63 hereinafter. The sequence of I(f) was derived as aconsensus sequence as described in Example 1 hereinbelow. Of compoundswithin formula I(f), those within the formula

I(f') (SEQ ID NO: 125) f¹f²f³KWDf⁷L⁹KQf¹²f¹³f¹⁴are preferred. Compounds falling within formula I(f) include SEQ ID NOS:32, 58, 60, 62, 63, 66, 67, 69, 70, 114, 115, 122, 123, 124, 147-150,152-177, 179, 180, 187.

Also in accordance with the present invention are compounds having theconsensus motif:

(SEQ ID NO: 110) PFPWEwhich also bind TALL-1.

Further in accordance with the present invention are compounds of theformulae:

I(g) (SEQ. ID. NO. 101) g¹g²g³Cg⁵PFg⁸Wg¹⁰Cg¹¹g¹²g¹³wherein:

-   -   g¹, g² and g³ are each independently absent or amino acid        residues;    -   g⁵ is a neutral hydrophobic residue;    -   g⁸ is a neutral hydrophobic residue;    -   g¹⁰ is an acidic residue;

I(h) (SEQ. ID. NO: 102) h¹h²h³CWh⁶h⁷WGh¹⁰Ch¹²h¹³h¹⁴wherein:

-   -   h¹, h², and h³ are each independently absent or amino acid        residues;    -   h⁶ is a hydrophobic residue;    -   h⁷ is a hydrophobic residue;    -   h¹⁰ is an acidic or polar hydrophobic residue; and    -   h¹², h¹³, and h¹⁴ are each independently absent or amino acid        residues.

I(i) (SEQ. ID. NO: 103) i¹i²i³Ci⁵i⁶i⁷i⁸i⁹i¹⁰Ci¹²i¹³i¹⁴wherein:

-   -   i¹ is absent or is an amino acid residue;    -   i² is a neutral hydrophobic residue;    -   i³ is an amino acid residue;    -   i⁵, i⁶, i⁷, and i⁸ are each independently amino acid residues;    -   i⁹ is an acidic residue;    -   i¹⁰ is an amino acid residue;    -   i¹² and i¹³ are each independently amino acid residues; and    -   i¹⁴ is a neutral hydrophobic residue.

The compounds defined by formulae I(g) through I(i) also bind TALL-1.

Further in accordance with present invention, modulators of TALL-1comprise:

-   -   a) a TALL-1 modulating domain (e.g., an amino acid sequence of        Formulae I(a) through I(i)), preferably the amino acid sequence        Dz²Lz⁴, or sequences derived therefrom by phage display,        RNA-peptide screening, or the other techniques mentioned above;        and    -   b) a vehicle, such as a polymer (e.g., PEG or dextran) or an Fc        domain, which is preferred;        wherein the vehicle is covalently attached to the TALL-1        modulating domain. The vehicle and the TALL-1 modulating domain        may be linked through the N- or C-terminus of the TALL-1        modulating domain, as described further below. The preferred        vehicle is an Fc domain, and the preferred Fc domain is an IgG        Fc domain. Such Fc-linked peptides are referred to herein as        “peptibodies.” Preferred TALL-1 modulating domains comprise the        amino acid sequences described hereinafter in Tables 1 and 2.        Other TALL-1 modulating domains can be generated by phage        display, RNA-peptide screening and the other techniques        mentioned herein.

Further in accordance with the present invention is a process for makingTALL-1 modulators, which comprises:

-   -   a. selecting at least one peptide that binds to TALL-1; and    -   b. covalently linking said peptide to a vehicle.        The preferred vehicle is an Fc domain. Step (a) is preferably        carried out by selection from the peptide sequences in Table 2        hereinafter or from phage display, RNA-peptide screening, or the        other techniques mentioned herein.

The compounds of this invention may be prepared by standard syntheticmethods, recombinant DNA techniques, or any other methods of preparingpeptides and fusion proteins. Compounds of this invention that encompassnon-peptide portions may be synthesized by standard organic chemistryreactions, in addition to standard peptide chemistry reactions whenapplicable.

The primary use contemplated for the compounds of this invention is astherapeutic or prophylactic agents. The vehicle-linked peptide may haveactivity comparable to—or even greater than—the natural ligand mimickedby the peptide.

The compounds of this invention may be used for therapeutic orprophylactic purposes by formulating them with appropriatepharmaceutical carrier materials and administering an effective amountto a patient, such as a human (or other mammal) in need thereof. Otherrelated aspects are also included in the instant invention.

Numerous additional aspects and advantages of the present invention willbecome apparent upon consideration of the figures and detaileddescription of the invention.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1A shows a single disulfide-bonded dimer with the Fc domain linkedat the amino terminus of the peptides or peptide-linker combinations ofX² as defined below. FIG. 1B shows a doubly disulfide bonded dimer withthe Fc domain linked at the amino terminus of the peptides orpeptide-linker combinations of X² as defined below. FIG. 1C shows anoncovalent dimer. FIG. 1D shows a single disulfide-bonded dimer withthe Fc domain linked at the carboxyl terminus of the peptides orpeptide-linker combinations of X¹ as defined below. FIG. 1E shows adouble disulfide bonded dimer with the Fc domain linked at the carboxylterminus of the peptides or peptide-linker combinations of X¹ as definedbelow. FIG. 1F shows a noncovalent dimer. IgG1 antibodies typically havetwo disulfide bonds at the hinge region of the antibody. The Fc domainin FIGS. 1A and 1D may be formed by truncation between the two disulfidebond sites or by substitution of a cysteinyl residue with an unreactiveresidue (e.g., alanyl). The Fc domain in FIGS. 1B and 1E may be formedby truncation of the parent antibody to retain both cysteinyl residuesin the Fc domain chains or by expression from a construct including asequence encoding such an Fc domain. The Fc domain in FIGS. 1C and 1Fmay be formed by elimination of the cysteinyl residues by eithertruncation or substitution. One may desire to eliminate the cysteinylresidues to avoid impurities formed by reaction of the cysteinyl residuewith cysteinyl residues of other proteins present in the host cell. Thenoncovalent bonding of the Fc domains is sufficient to hold together thedimer. Other dimers may be formed by using Fc domains derived fromdifferent types of antibodies (e.g., IgG2, IgM).

FIG. 2A shows the structure of a single chain molecule featuring tandemrepeats of the pharmacologically peptides P¹ and P² as defined below,and may also represent the DNA construct for the molecule. FIG. 2B showsa dimer in which the linker-peptide portion is present on only one chainof the dimer. FIG. 2C shows a dimer having the peptide portion on bothchains. The dimer of FIG. 2C will form spontaneously in certain hostcells upon expression of a DNA construct encoding the single chain shownin FIG. 3A. In other host cells, the cells could be placed in conditionsfavoring formation of dimers or the dimers can be formed in vitro.

FIGS. 3A and B show exemplary nucleic acid and amino acid sequences (SEQID NOS: 1 and 2, respectively) of human IgG1 Fc that may be used in thisinvention. FIG. 3A shows nucleotides 1-360 and encoded amino acids1-120. FIG. 3B shows nucleotides 361-684 and encoded amino acids121-228.

FIGS. 4A-F show the nucleotide and amino acid sequences (SEQ ID NOS:3-26) of NdeI to SalI fragments encoding peptide and linker. FIG. 4Ashows the nucleotide and amino acid sequences of AGP3-8-1-a (SEQ IDNOs:3 and 4) and AGP3-8-2-a (SEQ ID NOs:5 and 6). FIG. 4B shows thenucleotide and amino acid sequences of AGP3-8-4-a (SEQ ID NOs:7 and 8)and AGP3-12-4-a (SEQ ID NOs:9 and 10). FIG. 4C shows the nucleotide andamino acid sequences of AGP3-12-3-a (SEQ ID NOs:11 and 12) andAGP3-12-5-a (SEQ ID NOs:13 and 14). FIG. 4D shows the nucleotide andamino acid sequences of AGP3-12-8-a (SEQ ID NOs:15 and 16) andAGP3-12-9-a (SEQ ID NOs:17 and 18). FIG. 4E shows the nucleotide andamino acid sequences of AGP3-12-10-a (SEQ ID NOs:19 and 20) andAGP3-12-11-a (SEQ ID NOs:21 and 22). FIG. 4F shows the nucleotide andamino acid sequences of AGP3-12-14-a (SEQ ID NOs:23 and 24) and AGP3consensus (SEQ ID NOs:25 and 26).

FIGS. 5A-M show the nucleotide sequence (SEQ ID NO: 28) ofpAMG21-RANK-Fc vector, which was used to construct Fc-linked moleculesof the present invention. FIG. 5A shows nucleotides 1-480, which includeenzyme restriction sites Pfl 1108I, GblII, and ScaI, promoter regionPcopB, and the coding region and corresponding encoded amino acidsequence for copB protein. FIG. 5B shows nucleotides 481-560, whichinclude enzyme restriction sites BmnI, DrdII, and DraIII, promoterregion PrepA, a binding site for copB, and the coding regions andcorresponding encoded amino acid sequences for copT and repA1. FIG. 5Cshows nucleotides 961-1500, which include enzyme restriction sitesBstBI, AceIII, and AflII. FIG. 5D shows nucleotides 1501-2220, whichinclude enzyme restriction site PflMI and the coding region andcorresponding encoded amino acid sequence for repA4. FIG. 5E showsnucleotides 2221-2760, which include enzyme restriction sites BglI,SfiI, BstEII, and BspLullI. FIG. 5F shows nucleotides 2761-3480, whichinclude enzyme restriction sites NspV and BplI and the coding region andcorresponding encoded amino acid sequence for APHII. FIG. 5G showsnucleotides 3481-4080, which include enzyme restriction sites EagI andBcgI, promoter region APHII, APHII mRNA, and the coding region andcorresponding encoded amino acid sequence for APHII. FIG. 5H showsnucleotides 4081-4620, which include enzyme restriction sites NsiI,BsaI, and Psp1406I and T1 and T2 hairpins. FIG. 5I shows nucleotides4621-5220, which include enzyme restriction sites AatII and BsmI and thecoding region and corresponding encoded amino acid sequence for luxR.FIG. 5J shows nucleotides 5221-5760, which include enzyme restrictionsites NruI, ClaI, BbaI, and NdeI, the coding region and correspondingamino acid sequence of luxR and RANK, promoter regions luxPL and luxPR,and a CRP binding site. FIG. 5K shows nucleotides 5761-6240, whichinclude enzyme restriction sites ApaLI, Acc65I, KpnI, SalI, and AccI,and the coding region and corresponding amino acid sequence for Fc. FIG.5L shows nucleotides 6241-6780, which include enzyme restriction sitesBspEI, AhdI, BspHI, EconI, BsrGI, BmaI, SmaI, and SexAI. FIG. 5M showsnucleotides 6781-7285, which include enzyme restriction sites BamHI andBlpI and T7 and toop hairpins.

FIGS. 6A and B show the DNA sequence (SEQ ID NO: 97) inserted intopCFM1656 between the unique AatII (position #4364 in pCFM1656) and SacII(position #4585 in pCFM1656) restriction sites to form expressionplasmid pAMG21 (ATCC accession no. 98113). FIG. 6A shows the first partof the DNA sequence. FIG. 6B shows the second part of the DNA sequence.

FIG. 7 shows that the TALL-1 peptibody (SEQ ID NO: 70) inhibitsTALL-1-mediated B cell proliferation. Purified B cells (10⁵) from B6mice were cultured in triplicates in 96-well plated with the indicatedamounts of TALL-1 consensus peptibody in the presence of 10 ng/ml TALL-1plus 2 μg/ml anti-IgM antibody. Proliferation was measured byradioactive [³H]thymidine uptake in the last 18 h of pulse. Data shownrepresent mean±SD triplicate wells.

FIG. 8 shows that a TALL-1 N-terminal tandem dimer peptibodies (SEQ IDNO: 123, 124 in Table 5B hereinafter) are preferable for inhibition ofTALL-1-mediated B cell proliferation. Purified B cells (10⁵) from B6mice were cultured in triplicates in 96-well plated with the indicatedamounts of TALL-1 12-3 peptibody and TALL-1 consensus peptibody (SEQ IDNOS: 115 and 122 of Table 5B) or the related dimer peptibodies (SEQ IDNOS: 123, 124) in the presence of 10 μg/ml TALL-1 plus 2 μg/ml anti-IgMantibody. Proliferation was measured by radioactive [³H]thymidine uptakein the last 18 h of pulse. Data shown represent mean±SD triplicatewells.

FIG. 9 shows that AGP3 peptibody binds to AGP3 with high affinity.Dissociation equilibrium constant (K_(D)) was obtained from nonlinearregression of the competition curves using a dual-curve one-sitehomogeneous binding model (KinEx™ software). K_(D) is about 4 pM forAGP3 peptibody binding with human AGP3 (SEQ ID NO: 123).

FIGS. 10A and B show that AGP3 peptibody blocks both human and murineAGP3 in the Biacore competition assay. Soluble human TACI protein wasimmobilized to B1 chip. 1 nM of recombinant human AGP3 protein (FIG.10A) or 5 nM of recombinant murine AGP3 protein (FIG. 10B) was incubatedwith indicated amount of AGP3 peptibody before injected over the surfaceof receptor. Relative human AGP3 and murine AGP3 (binding response wasshown (SEQ ID NO: 123). FIG. 10A shows results for human AGP3. FIG. 10Bshows results for murine AGP3.

FIG. 11A shows that AGP3 peptibody blocked AGP3 binding to all threereceptors TACI, BCMA and BAFFR in Biacore competition assay. Recombinantsoluble receptor TACI, BCMA and BAFFR proteins were immobilized to CM5chip. 1 nM of recombinant human AGP3 (upper panel) were incubated withindicated amount of AGP3 peptibody before injected over each receptorsurface. Relative binding of AGP3 was measured. Similarly, 1 nM ofrecombinant APRIL protein was incubated with indicated amount of AGP3peptibody before injected over each receptor surface. FIG. 11B showsthat AGP3 peptibody didn't inhibit APRIL binding to all three receptors(SEQ ID NO: 123).

FIGS. 12A and B show that AGP3 peptibody inhibits mouse serumimmunoglobulin level increase induced by human AGP3 challenge. Balb/cmice received 7 daily intraperitoneal injections of 1 mg/Kg human AGP3protein along with saline, human Fc, or AGP3 peptibody at indicateddoses, and were bled on day 8. Serum total IgM and IgA level weremeasured by ELISA (SEQ ID NO: 123). FIG. 12A shows IgM levels. FIG. 12Bshows IgA levels.

FIG. 13 shows that AGP3 peptibody treatment reduced arthritis severityin the mouse CIA model. Eight to 12 weeks old DBA/1 male mice wereimmunized with bovine collagen type II (bCII) emulsified in completefreunds adjuvant intradermally at the base of tail, and were boosted 3weeks after the initial immunization with bCII emulsified in incompletefreunds adjuvant. Treatment with indicated dosage of AGP3 peptibody wasbegun from the day of booster immunization for 4 weeks. As describedbefore (Khare et al., J. Immunol. 155: 3653-9, 1995), all four paws wereindividually scored from 0-3 for arthritis severity (SEQ ID NO: 123).

FIG. 14 shows that AGP3 peptibody treatment inhibited anti-collagenantibody generation in the mouse CIA model. Serum samples were taken oneweek after final treatment (day 35) as described above. Serumanti-collagen II antibody level was determined by ELISA analysis (SEQ IDNO: 123).

FIGS. 15A and B show that AGP3 peptibody treatment delayed proteinuriaonset and improved survival in NZB/NZW lupus mice. Five-month-old lupusprone NZBx NZBWF1 mice were treated i.p. 3×/week for 8 weeks with PBS orindicated doses of AGP3 peptibody (SEQ ID NO: 123) or human Fc proteins.Protein in the urine was evaluated monthly throughout the life of theexperiment with Albustix reagent strips (Bayer AG). FIG. 15A showsdelayed proteinuria onset. FIG. 15B shows prolonged survival.

FIG. 16A shows the nucleic acid and amino acid sequences of a preferredTALL-1-binding peptibody (SEQ ID NOS: 189 and 123). FIG. 16A showsnucleotides 1-480 and amino acids 1-160. FIG. 16B shows nucleotides481-882 and amino acids 161-293.

DETAILED DESCRIPTION OF THE INVENTION Definition of Terms

The terms used throughout this specification are defined as follows,unless otherwise limited in specific instances.

General Definitions

The term “comprising” means that a compound may include additional aminoacids on either or both of the N- or C-termini of the given sequence. Ofcourse, these additional amino acids should not significantly interferewith the activity of the compound.

Additionally, physiologically acceptable salts of the compounds of thisinvention are also encompassed herein. The term “physiologicallyacceptable salts” refers to any salts that are known or later discoveredto be pharmaceutically acceptable. Some specific examples are: acetate;trifluoroacetate; hydrohalides, such as hydrochloride and hydrobromide;sulfate; citrate; tartrate; glycolate; and oxalate.

Amino Acids

The term “acidic residue” refers to amino acid residues in D- or L-formhaving sidechains comprising acidic groups. Exemplary acidic residuesinclude D and E.

The term “amide residue” refers to amino acids in D- or L-form havingsidechains comprising amide derivatives of acidic groups. Exemplaryresidues include N and Q.

The term “aromatic residue” refers to amino acid residues in D- orL-form having sidechains comprising aromatic groups. Exemplary aromaticresidues include F, Y, and W.

The term “basic residue” refers to amino acid residues in D- or L-formhaving sidechains comprising basic groups. Exemplary basic residuesinclude H, K, and R.

The term “hydrophilic residue” refers to amino acid residues in D- orL-form having sidechains comprising polar groups. Exemplary hydrophilicresidues include C, S, T, N, and Q.

The term “nonfunctional residue” refers to amino acid residues in D- orL-form having sidechains that lack acidic, basic, or aromatic groups.Exemplary nonfunctional amino acid residues include M, G, A, V, I, L andnorleucine (Nle).

The term “neutral hydrophobic residue” refers to amino acid residues inD- or L-form having sidechains that lack basic, acidic, or polar groups.Exemplary neutral hydrophobic amino acid residues include A, V, L, I, P,W, M, and F.

The term “polar hydrophobic residue” refers to amino acid residues in D-or L-form having sidechains comprising polar groups. Exemplary polarhydrophobic amino acid residues include T, G, S, Y, C, Q, and N.

The term “hydrophobic residue” refers to amino acid residues in D- orL-form having sidechains that lack basic or acidic groups. Exemplaryhydrophobic amino acid residues include A, V, L, I, P, W, M, F, T, G, S,Y, C, Q, and N.

Peptides

The term “peptide” refers to molecules of 1 to 40 amino acids, withmolecules of 5 to 20 amino acids preferred. Exemplary peptides maycomprise the TALL-1 modulating domain of a naturally occurring moleculeor comprise randomized sequences.

The term “randomized” as used to refer to peptide sequences refers tofully random sequences (e.g., selected by phage display methods orRNA-peptide screening) and sequences in which one or more residues of anaturally occurring molecule is replaced by an amino acid residue notappearing in that position in the naturally occurring molecule.Exemplary methods for identifying peptide sequences include phagedisplay, E. coli display, ribosome display, RNA-peptide screening,chemical screening, and the like.

The term “TALL-1 modulating domain” refers to any amino acid sequencethat binds to the TALL-1 and comprises naturally occurring sequences orrandomized sequences. Exemplary TALL-1 modulating domains can beidentified or derived by phage display or other methods mentionedherein.

The term “TALL-1 antagonist” refers to a molecule that binds to theTALL-1 and increases or decreases one or more assay parameters oppositefrom the effect on those parameters by full length native TALL-1. Suchactivity can be determined, for example, by such assays as described inthe subsection entitled “Biological activity of AGP-3” in the Materials& Methods section of the patent application entitled, “TNF-RELATEDPROTEINS”, WO 00/47740, published Aug. 17, 2000.

Vehicles and Peptibodies

The term “vehicle” refers to a molecule that prevents degradation and/orincreases half-life, reduces toxicity, reduces immunogenicity, orincreases biological activity of a therapeutic protein. Exemplaryvehicles include an Fc domain (which is preferred) as well as a linearpolymer (e.g., polyethylene glycol (PEG), polylysine, dextran, etc.); abranched-chain polymer (see, for example, U.S. Pat. No. 4,289,872 toDenkenwalter et al., issued Sep. 15, 1981; U.S. Pat. No. 5,229,490 toTam, issued Jul. 20, 1993; WO 93/21259 by Frechet et al., published 28Oct. 1993); a lipid; a cholesterol group (such as a steroid); acarbohydrate or oligosaccharide (e.g., dextran); any natural orsynthetic protein, polypeptide or peptide that binds to a salvagereceptor; albumin, including human serum albumin (HSA), leucine zipperdomain, and other such proteins and protein fragments. Vehicles arefurther described hereinafter.

The term “native Fc” refers to molecule or sequence comprising thesequence of a non-antigen-binding fragment resulting from digestion ofwhole antibody, whether in monomeric or multimeric form. The originalimmunoglobulin source of the native Fc is preferably of human origin andmay be any of the immunoglobulin, although IgG1 and IgG2 are preferred.Native Fc's are made up of monomeric polypeptides that may be linkedinto dimeric or multimeric forms by covalent (i.e., disulfide bonds) andnon-covalent association. The number of intermolecular disulfide bondsbetween monomeric subunits of native Fc molecules ranges from 1 to 4depending on class (e.g., IgG, IgA, IgE) or subclass (e.g., IgG1, IgG2,IgG3, IgA1, IgGA2). One example of a native Fc is a disulfide-bondeddimer resulting from papain digestion of an IgG (see Ellison et al.(1982), Nucleic Acids Res. 10: 4071-9). The term “native Fc” as usedherein is generic to the monomeric, dimeric, and multimeric forms.

The term “Fc variant” refers to a molecule or sequence that is modifiedfrom a native Fc but still comprises a binding site for the salvagereceptor, FcRn. International applications WO 97/34631 (published 25Sep. 1997) and WO 96/32478 describe exemplary Fc variants, as well asinteraction with the salvage receptor, and are hereby incorporated byreference in their entirety. Thus, the term “Fc variant” comprises amolecule or sequence that is humanized from a non-human native Fc.Furthermore, a native Fc comprises sites that may be removed becausethey provide structural features or biological activity that are notrequired for the fusion molecules of the present invention. Thus, theterm “Fc variant” comprises a molecule or sequence that lacks one ormore native Fc sites or residues that affect or are involved in (1)disulfide bond formation, (2) incompatibility with a selected host cell(3) N-terminal heterogeneity upon expression in a selected host cell,(4) glycosylation, (5) interaction with complement, (6) binding to an Fcreceptor other than a salvage receptor, or (7) antibody-dependentcellular cytotoxicity (ADCC). Fc variants are described in furtherdetail hereinafter.

The term “Fe domain” encompasses native Fc and Fc variant molecules andsequences as defined above. As with Fc variants and native Fe's, theterm “Fc domain” includes molecules in monomeric or multimeric form,whether digested from whole antibody or produced by other means.

The term “multimer” as applied to Fc domains or molecules comprising Fcdomains refers to molecules having two or more polypeptide chainsassociated covalently, noncovalently, or by both covalent andnon-covalent interactions. IgG molecules typically form dimers; IgM,pentamers; IgD, dimers; and IgA, monomers, dimers, trimers, ortetramers. Multimers may be formed by exploiting the sequence andresulting activity of the native Ig source of the Fc or by derivatizing(as defined below) such a native Fc.

The term “dimer” as applied to Fc domains or molecules comprising Fcdomains refers to molecules having two polypeptide chains associatedcovalently or non-covalently. Thus, exemplary dimers within the scope ofthis invention are as shown in FIG. 1.

The terms “derivatizing” and “derivative” or “derivatized” compriseprocesses and resulting compounds respectively in which (1) the compoundhas a cyclic portion; for example, cross-linking between cysteinylresidues within the compound; (2) the compound is cross-linked or has across-linking site; for example, the compound has a cysteinyl residueand thus forms cross-linked dimers in culture or in vivo; (3) one ormore peptidyl linkage is replaced by a non-peptidyl linkage; (4) theN-terminus is replaced by —NRR¹, NRC(O)R¹, —NRC(O)OR¹, —NRS(O)₂R¹,—NHC(O)NHR, a succinimide group, or substituted or unsubstitutedbenzyloxycarbonyl-NH—, wherein R and R¹ and the ring substituents are asdefined hereinafter; (5) the C-terminus is replaced by —C(O)R² or —NR³R⁴wherein R², R³ and R⁴ are as defined hereinafter; and (6) compounds inwhich individual amino acid moieties are modified through treatment withagents capable of reacting with selected side chains or terminalresidues. Derivatives are further described hereinafter.

The terms “peptibody” and “peptibodies” refer to molecules comprising anFc domain and at least one peptide. Such peptibodies may be multimers ordimers or fragments thereof, and they may be derivatized. In the presentinvention, the molecules of formulae II through VI hereinafter arepeptibodies when VI is an Fc domain.

Structure of Compounds

In General.

The present inventors identified sequences capable of binding to andmodulating the biological activity of TALL-1. These sequences can bemodified through the techniques mentioned above by which one or moreamino acids may be changed while maintaining or even improving thebinding affinity of the peptide.

In the compositions of matter prepared in accordance with thisinvention, the peptide(s) may be attached to the vehicle through thepeptide's N-terminus or C-terminus. Any of these peptides may be linkedin tandem (i.e., sequentially), with or without linkers. Thus, thevehicle-peptide molecules of this invention may be described by thefollowing formula:

(X¹)_(a)—V¹—(X²)_(b)  II

wherein:

-   -   V¹ is a vehicle (preferably an Fc domain);    -   X¹ and X² are each independently selected from -(L¹)_(c)-P¹,        -(L¹)_(c)-P¹-(L²)_(d)-P², -(L¹)_(c)-P¹-(L²)_(d)-P²-(L³)_(e)-P³,        and -(L²)_(c)-P¹-(L²)_(d)-P²-(L³)_(e)-P³-(L⁴)_(f)-P⁴    -   P³ and P⁴ are each independently sequences of TALL-1 modulating        domains, such as those of Formulae I(a) through I(i);    -   L¹, L², L³, and L⁴ are each independently linkers; and a, b, c,        d, e, and f are each independently 0 or 1, provided that at        least one of a and b is 1.

Thus, compound II comprises preferred compounds of the formulae

X¹—V¹  III

and multimers thereof wherein V¹ is an Fc domain and is attached at theC-terminus of A¹;

V¹—X²  IV

and multimers thereof wherein V¹ is an Fc domain and is attached at theN-terminus of A²;

V¹-(L¹)_(c)P¹  V

and multimers thereof wherein V¹ is an Fc domain and is attached at theN-terminus of -(L¹)_(c)-P¹; and

V¹-(L¹)_(c)-P¹-(L²)_(d)-P²  VI

and multimers thereof wherein V¹ is an Fc domain and is attached at theN-terminus of -L¹-P¹-L²-P².

Peptides.

The peptides of this invention are useful as TALL-1 modulating peptidesor as TALL-1 modulating domains in the molecules of formulae II throughVI. Molecules of this invention comprising these peptide sequences maybe prepared by methods known in the art.

Preferred peptide sequences are those of the foregoing formulae I(a)having the substituents identified below.

TABLE 1 Preferred peptide substituents Formula I(a) a⁸ is T; a⁹ is abasic residue (K most preferred); and a¹² is a neutral hydrophobicresidue (F most preferred). Formula I(b) b³ is D, Q, or E; b⁶ is W or Y;b¹⁰ is T; b¹¹ is K or R; and b¹⁴ is V or L. Formula I(c) c⁹ is T; c¹⁰ isK or R; c¹³ is a I, L, or V; and c¹⁷ is A or L. Formula I(d) d¹³ is T.Formula I(e) e¹¹ is T. Formula I(f) f⁶ is T; f⁷ is K; and f¹⁰ is V.Formula I(g) g⁵ is W; g⁸ is P; g¹⁰ is E; and g¹³ is a basic residue.Formula I(h) h¹ is G; h⁶ is A; h⁷ is a neutral hydrophobic residue; andh¹⁰ is an acidic residue. Formula I(i) i² is W; and i¹⁴ is W.

Preferred peptide sequences appear in Table 2 below.

TABLE 2  Preferred TALL-1 modulating domains SEQ Sequence ID NO:PGTCFPFPWECTHA 29 WGACWPFPWECFKE 30 VPFCDLLTKHCFEA 31 GSRCKYKWDVLTKQCFHH32 LPGCKWDLLIKQWVCDPL 33 SADCYFDILTKSDVCTSS 34 SDDCMYDQLTRMFICSNL 35DLNCKYDELTYKEWCQFN 36 FHDCKYDLLTRQMVCHGL 37 RNHCFWDHLLKQDICPSP 38ANQCWWDSLTKKNVCEFF 39 YKGRQMWDILTRSWVVSL 126 QDVGLWWDILTRAWMPNI 127QNAQRVWDLLIRTWVYPQ 128 GWNEAWWDELTKIWVLEQ 129 RITCDTWDSLIKKCVPQS 130GAIMQFWDSLTKTWLRQS 131 WLHSGWWDPLTKHWLQKV 132 SEWFFWFDPLTRAQLKFR 133GVWFWWFDPLTKQWTQAG 134 MQCKGYYDILTKWCVTNG 135 LWSKEVWDILTKSWVSQA 136KAAGWWFDWLTKVWVPAP 137 AYQTWFWDSLTRLWLSTT 138 SGQHFWWDLLTRSWTPST 139LGVGQKWDPLTKQWVSRG 140 VGKMCQWDPLIKRTVCVG 141 CRQGAKFDLLTKQCLLGR 142GQAIRHWDVLTKQWVDSQ 143 RGPCGSWDLLTKHCLDSQ 144 WQWKQQWDLLTKQMVWVG 145PITICRKDLLTKQVVCLD 146 KTCNGKWDLLTKQCLQQA 147 KCLKGKWDLLTKQCVTEV 148RCWNGKWDLLTKQCIHPW 149 NRDMRKWDPLIKQWIVRP 150 QAAAATWDLLTKQWLVPP 151PEGGPKWDPLTKQFLPPV 152 QTPQKKWDLLTKQWFTRN 153 IGSPCKWDLLTKQMICQT 154CTAAGKWDLLTKQCIQEK 155 VSQCMKWDLLTKQCLQGW 156 VWGTWKWDLLTKQYLPPQ 157GWWEMKWDLLTKQWYRPQ 158 TAQVSKWDLLTKQWLPLA 159 QLWGTKWDLLTKQYIQIM 160WATSQKWDLLTKQWVQNM 161 QRQCAKWDLLTKQCVLFY 162 KTTDCKWDLLTKQRICQV 163LLCQGKWDLLTKQCLKLR 164 LMWFWKWDLLTKQLVPTF 165 QTWAWKWDLLTKQWIGPM 166NKELLKWDLLTKQCRGRS 167 GQKDLKWDLLTKQYVRQS 168 PKPCQKWDLLTKQCLGSV 169GQIGWKWDLLTKQWIQTR 170 VWLDWKWDLLTKQWIHPQ 171 QEWEYKWDLLTKQWGWLR 172HWDSWKWDLLTKQWVVQA 173 TRPLQKWDLLTKQWLRVG 174 SDQWQKWDLLTKQWFWDV 175QQTFMKWDLLTKQWIRRH 176 QGECRKWDLLTKQCFPGQ 177 GQMGWRWDPLIKMCLGPS 178QLDGCKWDLLTKQKVCIP 179 HGYWQKWDLLTKQWVSSE 180 HQGQCGWDLLTRIYLPCH 181LHKACKWDLLTKQCWPMQ 182 GPPGSVWDLLTKIWIQTG 183 ITQDWRFDTLTRLWLPLR 184QGGFAAWDVLTKMWITVP 185 GHGTPWWDALTRIWILGV 186 VWPWQKWDLLTKQFVFQD 187WQWSWKWDLLTRQYISSS 188 NQTLWKWDLLTKQFITYM 60 PVYQGWWDTLTKLYIWDG 61WLDGGWRDPLIKRSVQLG 62 GHQQFKWDLLTKQWVQSN 63 QRVGQFWDVLTKMFITGS 64QAQGWSYDALIKIWIRWP 65 GWMHWKWDPLTKQALPWM 66 GHPTYKWDLLTKQWILQM 67WNNWSLWDPLTKLWLQQN 68 WQWGWKWDLLTKQWVQQQ 69 GQMGWRWDPLTKMWLGTS 70

It is noted that the known receptors for TALL-1 bear some sequencehomology with preferred peptides:

12-3                        LPGCKWDLLIKQWVCDPL BAFFR   MRRGPRSLRGRDAPVPTPCVPTECYDLLVRKCVDCRLL TACITICNHQSQRTCAAFCRSLSCRKEQGKFYDHLLRDCISCASI BCMAFVSPSQEIRGRFRRMLQMAGQCSQNEYFDSLLHACPCQLRC(SEQ ID NOS: 33, 195, 196, and 197, respectively).

Any peptide containing a cysteinyl residue may be cross-linked withanother Cys-containing peptide, either or both of which may be linked toa vehicle. Any peptide having more than one Cys residue may form anintrapeptide disulfide bond, as well. Any of these peptides may bederivatized as described hereinafter.

Additional useful peptide sequences may result from conservative and/ornon-conservative modifications of the amino acid sequences of thesequences in Table 2.

Conservative modifications will produce peptides having functional andchemical characteristics similar to those of the peptide from which suchmodifications are made. In contrast, substantial modifications in thefunctional and/or chemical characteristics of the peptides may beaccomplished by selecting substitutions in the amino acid sequence thatdiffer significantly in their effect on maintaining (a) the structure ofthe molecular backbone in the area of the substitution, for example, asa sheet or helical conformation, (b) the charge or hydrophobicity of themolecule at the target site, or (c) the size of the molecule.

For example, a “conservative amino acid substitution” may involve asubstitution of a native amino acid residue with a nonnative residuesuch that there is little or no effect on the polarity or charge of theamino acid residue at that position. Furthermore, any native residue inthe polypeptide may also be substituted with alanine, as has beenpreviously described for “alanine scanning mutagenesis” (see, forexample, MacLennan et al., 1998, Acta Physiol. Scand. Suppl. 643:55-67;Sasaki et al., 1998, Adv. Biophys. 35:1-24, which discuss alaninescanning mutagenesis).

Desired amino acid substitutions (whether conservative ornon-conservative) can be determined by those skilled in the art at thetime such substitutions are desired. For example, amino acidsubstitutions can be used to identify important residues of the peptidesequence, or to increase or decrease the affinity of the peptide orvehicle-peptide molecules (see preceding formulae) described herein.Exemplary amino acid substitutions are set forth in Table 3.

TABLE 3 Amino Acid Substitutions Original Exemplary Preferred ResiduesSubstitutions Substitutions Ala (A) Val, Leu, Ile Val Arg (R) Lys, Gln,Asn Lys Asn (N) Gln Gln Asp (D) Glu Glu Cys (C) Ser, Ala Ser Gln (Q) AsnAsn Glu (E) Asp Asp Gly (G) Pro, Ala Ala His (H) Asn, Gln, Lys, Arg ArgIle (I) Leu, Val, Met, Ala, Phe, Leu Norleucine Leu (L) Norleucine, Ile,Val, Met, Ile Ala, Phe Lys (K) Arg, 1,4 Diamino-butyric Arg Acid, Gln,Asn Met (M) Leu, Phe, Ile Leu Phe (F) Leu, Val, Ile, Ala, Tyr Leu Pro(P) Ala Gly Ser (S) Thr, Ala, Cys Thr Thr (T) Ser Ser Trp (W) Tyr, PheTyr Tyr (Y) Trp, Phe, Thr, Ser Phe Val (V) Ile, Met, Leu, Phe, Ala, LeuNorleucine

In certain embodiments, conservative amino acid substitutions alsoencompass non-naturally occurring amino acid residues which aretypically incorporated by chemical peptide synthesis rather than bysynthesis in biological systems.

As noted in the foregoing section “Definition of Terms,” naturallyoccurring residues may be divided into classes based on common sidechainproperties that may be useful for modifications of sequence. Forexample, non-conservative substitutions may involve the exchange of amember of one of these classes for a member from another class. Suchsubstituted residues may be introduced into regions of the peptide thatare homologous with non-human orthologs, or into the non-homologousregions of the molecule. In addition, one may also make modificationsusing P or G for the purpose of influencing chain orientation.

In making such modifications, the hydropathic index of amino acids maybe considered. Each amino acid has been assigned a hydropathic index onthe basis of their hydrophobicity and charge characteristics, these are:isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8);cysteine/cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine(−0.4); threonine (−0.7); serine (−0.8); tryptophan (−0.9); tyrosine(−1.3); proline (−1.6); histidine (−3.2); glutamate (−3.5); glutamine(−3.5); aspartate (−3.5); asparagine (−3.5); lysine (−3.9); and arginine(−4.5).

The importance of the hydropathic amino acid index in conferringinteractive biological function on a protein is understood in the art.Kyte et al., J. Mol. Biol., 157: 105-131 (1982). It is known thatcertain amino acids may be substituted for other amino acids having asimilar hydropathic index or score and still retain a similar biologicalactivity. In making changes based upon the hydropathic index, thesubstitution of amino acids whose hydropathic indices are within ±2 ispreferred, those which are within ±1 are particularly preferred, andthose within ±0.5 are even more particularly preferred.

It is also understood in the art that the substitution of like aminoacids can be made effectively on the basis of hydrophilicity. Thegreatest local average hydrophilicity of a protein, as governed by thehydrophilicity of its adjacent amino acids, correlates with itsimmunogenicity and antigenicity, i.e. a biological property of theprotein.

The following hydrophilicity values have been assigned to amino acidresidues: arginine (+3.0); lysine (+3.0); aspartate (+3.0±1); glutamate(+3.0±1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine(0); threonine (−0.4); proline (−0.5±1); alanine (−0.5); histidine(−0.5); cysteine (−1.0); methionine (−1.3); valine (−1.5); leucine(−1.8); isoleucine (−1.8); tyrosine (−2.3); phenylalanine (−2.5);tryptophan (−3.4). In making changes based upon similar hydrophilicityvalues, the substitution of amino acids whose hydrophilicity values arewithin ±2 is preferred, those which are within ±1 are particularlypreferred, and those within ±0.5 are even more particularly preferred.One may also identify epitopes from primary amino acid sequences on thebasis of hydrophilicity. These regions are also referred to as “epitopiccore regions.”

A skilled artisan will be able to determine suitable variants of thepolypeptide as set forth in the foregoing sequences using well knowntechniques. For identifying suitable areas of the molecule that may bechanged without destroying activity, one skilled in the art may targetareas not believed to be important for activity. For example, whensimilar polypeptides with similar activities from the same species orfrom other species are known, one skilled in the art may compare theamino acid sequence of a peptide to similar peptides. With such acomparison, one can identify residues and portions of the molecules thatare conserved among similar polypeptides. It will be appreciated thatchanges in areas of a peptide that are not conserved relative to suchsimilar peptides would be less likely to adversely affect the biologicalactivity and/or structure of the peptide. One skilled in the art wouldalso know that, even in relatively conserved regions, one may substitutechemically similar amino acids for the naturally occurring residueswhile retaining activity (conservative amino acid residuesubstitutions). Therefore, even areas that may be important forbiological activity or for structure may be subject to conservativeamino acid substitutions without destroying the biological activity orwithout adversely affecting the peptide structure.

Additionally, one skilled in the art can review structure-functionstudies identifying residues in similar peptides that are important foractivity or structure. In view of such a comparison, one can predict theimportance of amino acid residues in a peptide that correspond to aminoacid residues that are important for activity or structure in similarpeptides. One skilled in the art may opt for chemically similar aminoacid substitutions for such predicted important amino acid residues ofthe peptides.

One skilled in the art can also analyze the three-dimensional structureand amino acid sequence in relation to that structure in similarpolypeptides. In view of that information, one skilled in the art maypredict the alignment of amino acid residues of a peptide with respectto its three dimensional structure. One skilled in the art may choosenot to make radical changes to amino acid residues predicted to be onthe surface of the protein, since such residues may be involved inimportant interactions with other molecules. Moreover, one skilled inthe art may generate test variants containing a single amino acidsubstitution at each desired amino acid residue. The variants can thenbe screened using activity assays know to those skilled in the art. Suchdata could be used to gather information about suitable variants. Forexample, if one discovered that a change to a particular amino acidresidue resulted in destroyed, undesirably reduced, or unsuitableactivity, variants with such a change would be avoided. In other words,based on information gathered from such routine experiments, one skilledin the art can readily determine the amino acids where furthersubstitutions should be avoided either alone or in combination withother mutations.

A number of scientific publications have been devoted to the predictionof secondary structure. See Moult J., Curr. Op. in Biotech., 7(4):422-427 (1996), Chou et al., Biochemistry, 13(2): 222-245 (1974); Chouet al., Biochemistry, 113(2): 211-222 (1974); Chou et al., Adv. Enzymol.Relat. Areas Mol. Biol., 47: 45-148 (1978); Chou et al., Ann. Rev.Biochem., 47: 251-276 and Chou et al., Biophys. J., 26: 367-384 (1979).Moreover, computer programs are currently available to assist withpredicting secondary structure. One method of predicting secondarystructure is based upon homology modeling. For example, two polypeptidesor proteins which have a sequence identity of greater than 30%, orsimilarity greater than 40% often have similar structural topologies.The recent growth of the protein structural data base (PDB) has providedenhanced predictability of secondary structure, including the potentialnumber of folds within a polypeptide's or protein's structure. See Holmet al., Nucl. Acid. Res., 27(1): 244-247 (1999). It has been suggested(Brenner et al., Curr. Op. Struct. Biol., 7(3): 369-376 (1997)) thatthere are a limited number of folds in a given polypeptide or proteinand that once a critical number of structures have been resolved,structural prediction will gain dramatically in accuracy.

Additional methods of predicting secondary structure include “threading”(Jones, D., Curr. Opin. Struct. Biol., 7(3): 377-87 (1997); Sippl etal., Structure, 4(1): 15-9 (1996)), “profile analysis” (Bowie et al.,Science, 253: 164-170 (1991); Gribskov et al., Meth. Enzym., 183:146-159 (1990); Gribskov et al., Proc. Nat. Acad. Sci., 84(13): 4355-8(1987)), and “evolutionary linkage” (See Home, supra, and Brenner,supra).

Vehicles.

This invention requires the presence of at least one vehicle (V¹)attached to a peptide through the N-terminus, C-terminus or a sidechainof one of the amino acid residues. Multiple vehicles may also be used;e.g., Fc's at each terminus or an Fc at a terminus and a PEG group atthe other terminus or a sidechain. Exemplary vehicles include:

-   -   an Fc domain;    -   other proteins, polypeptides, or peptides capable of binding to        a salvage receptor;    -   human serum albumin (HSA);    -   a leucine zipper (LZ) domain;    -   polyethylene glycol (PEG), including 5 kD, 20 kD, and 30 kD PEG,        as well as other polymers;    -   dextran;        and other molecules known in the art to provide extended        half-life and/or protection from proteolytic degradation or        clearance.

An Fc domain is the preferred vehicle. The Fc domain may be fused to theN or C termini of the peptides or at both the N and C termini. Fusion tothe N terminus is preferred.

As noted above, Fc variants are suitable vehicles within the scope ofthis invention. A native Fc may be extensively modified to form an Fcvariant in accordance with this invention, provided binding to thesalvage receptor is maintained; see, for example WO 97/34631 and WO96/32478. In such Fc variants, one may remove one or more sites of anative Fc that provide structural features or functional activity notrequired by the fusion molecules of this invention. One may remove thesesites by, for example, substituting or deleting residues, insertingresidues into the site, or truncating portions containing the site. Theinserted or substituted residues may also be altered amino acids, suchas peptidomimetics or D-amino acids. Fc variants may be desirable for anumber of reasons, several of which are described below. Exemplary Fcvariants include molecules and sequences in which:

-   1. Sites involved in disulfide bond formation are removed. Such    removal may avoid reaction with other cysteine-containing proteins    present in the host cell used to produce the molecules of the    invention. For this purpose, the cysteine-containing segment at the    N-terminus may be truncated or cysteine residues may be deleted or    substituted with other amino acids (e.g., alanyl, seryl). In    particular, one may truncate the N-terminal 20-amino acid segment of    SEQ ID NO: 2 or delete or substitute the cysteine residues at    positions 7 and 10 of SEQ ID NO: 2. Even when cysteine residues are    removed, the single chain Fc domains can still form a dimeric Fc    domain that is held together non-covalently.-   2. A native Fc is modified to make it more compatible with a    selected host cell. For example, one may remove the PA sequence near    the N-terminus of a typical native Fc, which may be recognized by a    digestive enzyme in E. coli such as proline iminopeptidase. One may    also add an N-terminal methionine residue, especially when the    molecule is expressed recombinantly in a bacterial cell such as E.    coli. The Fc domain of SEQ ID NO: 2 is one such Fc variant.-   3. A portion of the N-terminus of a native Fc is removed to prevent    N-terminal heterogeneity when expressed in a selected host cell. For    this purpose, one may delete any of the first 20 amino acid residues    at the N-terminus, particularly those at positions 1, 2, 3, 4 and 5.-   4. One or more glycosylation sites are removed. Residues that are    typically glycosylated (e.g., asparagine) may confer cytolytic    response. Such residues may be deleted or substituted with    unglycosylated residues (e.g., alanine).-   5. Sites involved in interaction with complement, such as the Clq    binding site, are removed. For example, one may delete or substitute    the EKK sequence of human IgG1. Complement recruitment may not be    advantageous for the molecules of this invention and so may be    avoided with such an Fc variant.-   6. Sites are removed that affect binding to Fc receptors other than    a salvage receptor. A native Fc may have sites for interaction with    certain white blood cells that are not required for the fusion    molecules of the present invention and so may be removed.-   7. The ADCC site is removed. ADCC sites are known in the art; see,    for example, Molec. Immunol. 29 (5): 633-9 (1992) with regard to    ADCC sites in IgG1. These sites, as well, are not required for the    fusion molecules of the present invention and so may be removed.-   8. When the native Fc is derived from a non-human antibody, the    native Fc may be humanized. Typically, to humanize a native Fc, one    will substitute selected residues in the non-human native Fc with    residues that are normally found in human native Fc. Techniques for    antibody humanization are well known in the art.

Preferred Fc variants include the following. In SEQ ID NO: 2 (FIGS. 3Aand B), the leucine at position 15 may be substituted with glutamate;the glutamate at position 99, with alanine; and the lysines at positions101 and 103, with alanines. In addition, one or more tyrosine residuescan be replaced by phenyalanine residues.

An alternative vehicle would be a protein, polypeptide, peptide,antibody, antibody fragment, or small molecule (e.g., a peptidomimeticcompound) capable of binding to a salvage receptor. For example, onecould use as a vehicle a polypeptide as described in U.S. Pat. No.5,739,277, issued Apr. 14, 1998 to Presta et al. Peptides could also beselected by phage display or RNA-peptide screening for binding to theFcRn salvage receptor. Such salvage receptor-binding compounds are alsoincluded within the meaning of “vehicle” and are within the scope ofthis invention. Such vehicles should be selected for increased half-life(e.g., by avoiding sequences recognized by proteases) and decreasedimmunogenicity (e.g., by favoring non-immunogenic sequences, asdiscovered in antibody humanization).

As noted above, polymer vehicles may also be used for V¹. Various meansfor attaching chemical moieties useful as vehicles are currentlyavailable, see, e.g., Patent Cooperation Treaty (“PCT”) InternationalPublication No. WO 96/11953, entitled “N-Terminally Chemically ModifiedProtein Compositions and Methods,” herein incorporated by reference inits entirety. This PCT publication discloses, among other things, theselective attachment of water soluble polymers to the N-terminus ofproteins.

A preferred polymer vehicle is polyethylene glycol (PEG). The PEG groupmay be of any convenient molecular weight and may be linear or branched.The average molecular weight of the PEG will preferably range from about2 kiloDalton (“kD”) to about 100 kD, more preferably from about 5 kD toabout 50 kD, most preferably from about 5 kD to about 10 kD. The PEGgroups will generally be attached to the compounds of the invention viaacylation or reductive alkylation through a reactive group on the PEGmoiety (e.g., an aldehyde, amino, thiol, or ester group) to a reactivegroup on the inventive compound (e.g., an aldehyde, amino, or estergroup).

A useful strategy for the PEGylation of synthetic peptides consists ofcombining, through forming a conjugate linkage in solution, a peptideand a PEG moiety, each bearing a special functionality that is mutuallyreactive toward the other. The peptides can be easily prepared withconventional solid phase synthesis. The peptides are “preactivated” withan appropriate functional group at a specific site. The precursors arepurified and fully characterized prior to reacting with the PEG moiety.Ligation of the peptide with PEG usually takes place in aqueous phaseand can be easily monitored by reverse phase analytical HPLC. ThePEGylated peptides can be easily purified by preparative HPLC andcharacterized by analytical HPLC, amino acid analysis and laserdesorption mass spectrometry.

Polysaccharide polymers are another type of water soluble polymer whichmay be used for protein modification. Dextrans are polysaccharidepolymers comprised of individual subunits of glucose predominantlylinked by α1-6 linkages. The dextran itself is available in manymolecular weight ranges, and is readily available in molecular weightsfrom about 1 kD to about 70 kD. Dextran is a suitable water solublepolymer for use in the present invention as a vehicle by itself or incombination with another vehicle (e.g., Fc). See, for example, WO96/11953 and WO 96/05309. The use of dextran conjugated to therapeuticor diagnostic immunoglobulin has been reported; see, for example,European Patent Publication No. 0 315 456, which is hereby incorporatedby reference in its entirety. Dextran of about 1 kD to about 20 kD ispreferred when dextran is used as a vehicle in accordance with thepresent invention.

Linkers.

Any “linker” group is optional. When present, its chemical structure isnot critical, since it serves primarily as a spacer. The linker ispreferably made up of amino acids linked together by peptide bonds.Thus, in preferred embodiments, the linker is made up of from 1 to 30amino acids linked by peptide bonds, wherein the amino acids areselected from the 20 naturally occurring amino acids. Some of theseamino acids may be glycosylated, as is well understood by those in theart. In a more preferred embodiment, the 1 to 20 amino acids areselected from glycine, alanine, proline, asparagine, glutamine, andlysine. Even more preferably, a linker is made up of a majority of aminoacids that are sterically unhindered, such as glycine and alanine. Thus,preferred linkers are polyglycines (particularly (Gly)₄, (Gly)₅),poly(Gly-Ala), and polyalanines. Other specific examples of linkers are:

(SEQ ID NO: 40) (Gly)₃Lys(Gly)₄; (SEQ ID NO: 41) (Gly)₃AsnGlySer(Gly)₂;(SEQ ID NO: 42) (Gly)₃Cys(Gly)₄;  and (SEQ ID NO: 43) GlyProAsnGlyGly.To explain the above nomenclature, for example, (Gly)₃Lys(Gly)₄ meansGly-Gly-Gly-Lys-Gly-Gly-Gly-Gly (SEQ ID NO: 40). Combinations of Gly andAla are also preferred. The linkers shown here are exemplary; linkerswithin the scope of this invention may be much longer and may includeother residues.

Preferred linkers are amino acid linkers comprising greater than 5 aminoacids, with suitable linkers having up to about 500 amino acids selectedfrom glycine, alanine, proline, asparagine, glutamine, lysine,threonine, serine or aspartate. Linkers of about 20 to 50 amino acidsare most preferred. One group of preferred linkers are those of theformulae

(SEQ ID NO: 193) GSGSATGGSGSTASSGSGSATx¹x² and (SEQ ID NO: 194)GSGSATGGSGSTASSGSGSATx¹x²GSGSATGGSGSTASSGSGSATx³x⁴wherein x¹ and x³ are each independently basic or hydrophobic residuesand x² and x⁴ are each independently hydrophobic residues. Specificpreferred linkers are:

(SEQ ID NO: 59) GSGSATGGSGSTASSGSGSATHM (SEQ ID NO: 190)GSGSATGGSGSTASSGSGSATGM (SEQ ID NO: 191) GSGSATGGSGSTASSGSGSATGS, and(SEQ ID NO: 192) GSGSATGGSGSTASSGSGSATHMGSGSATGGSGSTASSGSGSATHM.

Non-peptide linkers are also possible. For example, alkyl linkers suchas —NH—(CH₂)_(s)—C(O)—, wherein s=2-20 could be used. These alkyllinkers may further be substituted by any non-sterically hindering groupsuch as lower alkyl (e.g., C₁-C₆) lower acyl, halogen (e.g., Cl, Br),CN, NH₂, phenyl, etc. An exemplary non-peptide linker is a PEG linker,

wherein n is such that the linker has a molecular weight of 100 to 5000kD, preferably 100 to 500 kD. The peptide linkers may be altered to formderivatives in the same manner as described above.

Derivatives.

The inventors also contemplate derivatizing the peptide and/or vehicleportion of the compounds. Such derivatives may improve the solubility,absorption, biological half life, and the like of the compounds. Themoieties may alternatively eliminate or attenuate any undesirableside-effect of the compounds and the like. Exemplary derivatives includecompounds in which:

-   1. The compound or some portion thereof is cyclic. For example, the    peptide portion may be modified to contain two or more Cys residues    (e.g., in the linker), which could cyclize by disulfide bond    formation.-   2. The compound is cross-linked or is rendered capable of    cross-linking between molecules. For example, the peptide portion    may be modified to contain one Cys residue and thereby be able to    form an intermolecular disulfide bond with a like molecule. The    compound may also be cross-linked through its C-terminus, as in the    molecule shown below.

-    In Formula VIII, each “V¹” may represent typically one strand of    the Fc domain.-   3. One or more peptidyl [—C(O)NR-] linkages (bonds) is replaced by a    non-peptidyl linkage. Exemplary non-peptidyl linkages are —CH₂—    carbamate [—CH₂—OC(O)NR—], phosphonate, —CH₂-sulfonamide    [—CH₂—S(O)₂NR-1, urea [—NHC(O)NH—], —CH₂-secondary amine, and    alkylated peptide [—C(O)NR⁶— wherein R⁶ is lower alkyl].-   4. The N-terminus is derivatized. Typically, the N-terminus may be    acylated or modified to a substituted amine. Exemplary N-terminal    derivative groups include —NRR¹ (other than —NH₂), —NRC(O)R¹,    —NRC(O)OR¹, —NRS(O)₂R¹, —NHC(O)NHR¹, succinimide, or    benzyloxycarbonyl-NH— (CBZ—NH—), wherein R and R¹ are each    independently hydrogen or lower alkyl and wherein the phenyl ring    may be substituted with 1 to 3 substituents selected from the group    consisting of C₁-C₄ alkyl, C₁-C₄ alkoxy, chloro, and bromo.-   5. The free C-terminus is derivatized. Typically, the C-terminus is    esterified or amidated. Exemplary C-terminal derivative groups    include, for example, —C(O)R² wherein R² is lower alkoxy or —NR³R⁴    wherein R³ and R⁴ are independently hydrogen or C₁-C₈ alkyl    (preferably C₁-C₄ alkyl).-   6. A disulfide bond is replaced with another, preferably more    stable, cross-linking moiety (e.g., an alkylene). See, e.g.,    Bhatnagar et al. (1996), J. Med. Chem. 39: 3814-9; Alberts et    al. (1993) Thirteenth Am. Pep. Symp., 357-9.-   7. One or more individual amino acid residues is modified. Various    derivatizing agents are known to react specifically with selected    sidechains or terminal residues, as described in detail below.

Lysinyl residues and amino terminal residues may be reacted withsuccinic or other carboxylic acid anhydrides, which reverse the chargeof the lysinyl residues. Other suitable reagents for derivatizingalpha-amino-containing residues include imidoesters such as methylpicolinimidate; pyridoxal phosphate; pyridoxal; chloroborohydride;trinitrobenzenesulfonic acid; O-methylisourea; 2,4 pentanedione; andtransaminase-catalyzed reaction with glyoxylate.

Arginyl residues may be modified by reaction with any one or combinationof several conventional reagents, including phenylglyoxal,2,3-butanedione, 1,2-cyclohexanedione, and ninhydrin. Derivatization ofarginyl residues requires that the reaction be performed in alkalineconditions because of the high pKa of the guanidine functional group.Furthermore, these reagents may react with the groups of lysine as wellas the arginine epsilon-amino group.

Specific modification of tyrosyl residues has been studied extensively,with particular interest in introducing spectral labels into tyrosylresidues by reaction with aromatic diazonium compounds ortetranitromethane. Most commonly, N-acetylimidizole andtetranitromethane are used to form 0-acetyl tyrosyl species and 3-nitroderivatives, respectively.

Carboxyl sidechain groups (aspartyl or glutamyl) may be selectivelymodified by reaction with carbodiimides (R—N═C═N—R) such as1-cyclohexyl-3-(2-morpholinyl-(4-ethyl) carbodiimide or1-ethyl-3-(4-azonia-4,4-dimethylpentyl) carbodiimide. Furthermore,aspartyl and glutamyl residues may be converted to asparaginyl andglutaminyl residues by reaction with ammonium ions.

Glutaminyl and asparaginyl residues may be deamidated to thecorresponding glutamyl and aspartyl residues. Alternatively, theseresidues are deamidated under mildly acidic conditions. Either form ofthese residues falls within the scope of this invention.

Cysteinyl residues can be replaced by amino acid residues or othermoieties either to eliminate disulfide bonding or, conversely, tostabilize cross-linking. See, e.g., Bhatnagar et al. (1996), J. Med.Chem. 39: 3814-9.

Derivatization with bifunctional agents is useful for cross-linking thepeptides or their functional derivatives to a water-insoluble supportmatrix or to other macromolecular vehicles. Commonly used cross-linkingagents include, e.g., 1,1-bis(diazoacetyl)-2-phenylethane,glutaraldehyde, N-hydroxysuccinimide esters, for example, esters with4-azidosalicylic acid, homobifunctional imidoesters, includingdisuccinimidyl esters such as 3,3′-dithiobis(succinimidylpropionate),and bifunctional maleimides such as bis-N-maleimido-1,8-octane.Derivatizing agents such asmethyl-3-[(p-azidophenyl)dithio]propioimidate yield photoactivatableintermediates that are capable of forming cross-links in the presence oflight. Alternatively, reactive water-insoluble matrices such as cyanogenbromide-activated carbohydrates and the reactive substrates described inU.S. Pat. Nos. 3,969,287; 3,691,016; 4,195,128; 4,247,642; 4,229,537;and 4,330,440 are employed for protein immobilization.

Carbohydrate (oligosaccharide) groups may conveniently be attached tosites that are known to be glycosylation sites in proteins. Generally,O-linked oligosaccharides are attached to serine (Ser) or threonine(Thr) residues while N-linked oligosaccharides are attached toasparagine (Asn) residues when they are part of the sequenceAsn-X-Ser/Thr, where X can be any amino acid except proline. X ispreferably one of the 19 naturally occurring amino acids other thanproline. The structures of N-linked and O-linked oligosaccharides andthe sugar residues found in each type are different. One type of sugarthat is commonly found on both is N-acetylneuraminic acid (referred toas sialic acid). Sialic acid is usually the terminal residue of bothN-linked and O-linked oligosaccharides and, by virtue of its negativecharge, may confer acidic properties to the glycosylated compound. Suchsite(s) may be incorporated in the linker of the compounds of thisinvention and are preferably glycosylated by a cell during recombinantproduction of the polypeptide compounds (e.g., in mammalian cells suchas CHO, BHK, COS). However, such sites may further be glycosylated bysynthetic or semi-synthetic procedures known in the art.

Other possible modifications include hydroxylation of proline andlysine, phosphorylation of hydroxyl groups of seryl or threonylresidues, oxidation of the sulfur atom in Cys, methylation of thealpha-amino groups of lysine, arginine, and histidine side chains.Creighton, Proteins: Structure and Molecule Properties (W. H. Freeman &Co., San Francisco), pp. 79-86 (1983).

Compounds of the present invention may be changed at the DNA level, aswell. The DNA sequence of any portion of the compound may be changed tocodons more compatible with the chosen host cell. For E. coli, which isthe preferred host cell, optimized codons are known in the art. Codonsmay be substituted to eliminate restriction sites or to include silentrestriction sites, which may aid in processing of the DNA in theselected host cell. The vehicle, linker and peptide DNA sequences may bemodified to include any of the foregoing sequence changes.

Methods of Making

The compounds of this invention largely may be made in transformed hostcells using recombinant DNA techniques. To do so, a recombinant DNAmolecule coding for the peptide is prepared. Methods of preparing suchDNA molecules are well known in the art. For instance, sequences codingfor the peptides could be excised from DNA using suitable restrictionenzymes. Alternatively, the DNA molecule could be synthesized usingchemical synthesis techniques, such as the phosphoramidate method. Also,a combination of these techniques could be used.

The invention also includes a vector capable of expressing the peptidesin an appropriate host. The vector comprises the DNA molecule that codesfor the peptides operatively linked to appropriate expression controlsequences. Methods of effecting this operative linking, either before orafter the DNA molecule is inserted into the vector, are well known.Expression control sequences include promoters, activators, enhancers,operators, ribosomal binding sites, start signals, stop signals, capsignals, polyadenylation signals, and other signals involved with thecontrol of transcription or translation.

The resulting vector having the DNA molecule thereon is used totransform an appropriate host. This transformation may be performedusing methods well known in the art.

Any of a large number of available and well-known host cells may be usedin the practice of this invention. The selection of a particular host isdependent upon a number of factors recognized by the art. These include,for example, compatibility with the chosen expression vector, toxicityof the peptides encoded by the DNA molecule, rate of transformation,ease of recovery of the peptides, expression characteristics, bio-safetyand costs. A balance of these factors must be struck with theunderstanding that not all hosts may be equally effective for theexpression of a particular DNA sequence. Within these generalguidelines, useful microbial hosts include bacteria (such as E. colisp.), yeast (such as Saccharomyces sp.) and other fungi, insects,plants, mammalian (including human) cells in culture, or other hostsknown in the art.

Next, the transformed host is cultured and purified. Host cells may becultured under conventional fermentation conditions so that the desiredcompounds are expressed. Such fermentation conditions are well known inthe art. Finally, the peptides are purified from culture by methods wellknown in the art.

The compounds may also be made by synthetic methods. For example, solidphase synthesis techniques may be used. Suitable techniques are wellknown in the art, and include those described in Merrifield (1973),Chem. Polypeptides, pp. 335-61 (Katsoyannis and Panayotis eds.);Merrifield (1963), J. Am. Chem. Soc. 85: 2149; Davis et al. (1985),Biochem. Intl. 10: 394-414; Stewart and Young (1969), Solid PhasePeptide Synthesis; U.S. Pat. No. 3,941,763; Finn et al. (1976), TheProteins (3rd ed.) 2: 105-253; and Erickson et al. (1976), The Proteins(3rd ed.) 2: 257-527. Solid phase synthesis is the preferred techniqueof making individual peptides since it is the most cost-effective methodof making small peptides. Compounds that contain derivatized peptides orwhich contain non-peptide groups may be synthesized by well-knownorganic chemistry techniques.

Uses of the Compounds

Compounds of this invention may be particularly useful in treatment ofB-cell mediated autoimmune diseases. In particular, the compounds ofthis invention may be useful in treating, preventing, ameliorating,diagnosing or prognosing lupus, including systemic lupus erythematosus(SLE), and lupus-associated diseases and conditions. Other preferredindications include B-cell mediated cancers, including B-cell lymphoma.

The compounds of this invention can also be used to treat inflammatoryconditions of the joints. Inflammatory conditions of a joint are chronicjoint diseases that afflict and disable, to varying degrees, millions ofpeople worldwide. Rheumatoid arthritis is a disease of articular jointsin which the cartilage and bone are slowly eroded away by aproliferative, invasive connective tissue called pannus, which isderived from the synovial membrane. The disease may involveperi-articular structures such as bursae, tendon sheaths and tendons aswell as extra-articular tissues such as the subcutis, cardiovascularsystem, lungs, spleen, lymph nodes, skeletal muscles, nervous system(central and peripheral) and eyes (Silberberg (1985), Anderson'sPathology, Kissane (ed.), 11:1828). Osteoarthritis is a common jointdisease characterized by degenerative changes in articular cartilage andreactive proliferation of bone and cartilage around the joint.Osteoarthritis is a cell-mediated active process that may result fromthe inappropriate response of chondrocytes to catabolic and anabolicstimuli. Changes in some matrix molecules of articular cartilagereportedly occur in early osteoarthritis (Thonar et al. (1993),Rheumatic disease clinics of North America, Moskowitz (ed.), 19:635-657and Shinmei et al. (1992), Arthritis Rheum., 35:1304-1308). TALL-1,TALL-1R and modulators thereof are believed to be useful in thetreatment of these and related conditions.

Compounds of this invention may also be useful in treatment of a numberof additional diseases and disorders, including:

-   -   acute pancreatitis;    -   ALS;    -   Alzheimer's disease;    -   asthma;    -   atherosclerosis;    -   autoimmune hemolytic anemia;    -   cancer, particularly cancers related to B cells;    -   cachexia/anorexia;    -   chronic fatigue syndrome;    -   cirrhosis (e.g., primary biliary cirrhosis);    -   diabetes (e.g., insulin diabetes);    -   fever;    -   glomerulonephritis, including IgA glomerulonephritis and primary        glomerulonephritis;    -   Goodpasture's syndrome;    -   Guillain-Barre syndrome;    -   graft versus host disease;    -   Hashimoto's thyroiditis;    -   hemorrhagic shock;    -   hyperalgesia;    -   inflammatory bowel disease;    -   inflammatory conditions of a joint, including osteoarthritis,        psoriatic arthritis and rheumatoid arthritis;    -   inflammatory conditions resulting from strain, sprain, cartilage        damage, trauma, orthopedic surgery, infection or other disease        processes;    -   insulin-dependent diabetes mellitus;    -   ischemic injury, including cerebral ischemia (e.g., brain injury        as a result of trauma, epilepsy, hemorrhage or stroke, each of        which may lead to neurodegeneration);    -   learning impairment;    -   lung diseases (e.g., ARDS);    -   multiple myeloma;    -   multiple sclerosis;    -   Myasthenia gravis;    -   myelogenous (e.g., AML and CIVIL) and other leukemias;    -   myopathies (e.g., muscle protein metabolism, esp. in sepsis);    -   neurotoxicity (e.g., as induced by HIV);    -   osteoporosis;    -   pain;    -   Parkinson's disease;    -   Pemphigus;    -   polymyositis/dermatomyositis;    -   pulmonary inflammation, including autoimmune pulmonary        inflammation;    -   pre-term labor;    -   psoriasis;    -   Reiter's disease;    -   reperfusion injury;    -   septic shock;    -   side effects from radiation therapy;    -   Sjogren's syndrome;    -   sleep disturbance;    -   temporal mandibular joint disease;    -   thrombocytopenia, including idiopathic thrombocytopenia and        autoimmune neonatal thrombocytopenia;    -   tumor metastasis;    -   uveitis; and    -   vasculitis.

Compounds of this invention may be administered alone or in combinationwith a therapeutically effective amount of other drugs, includinganalgesic agents, disease-modifying anti-rheumatic drugs (DMARDs),non-steroidal anti-inflammatory drugs (NSAIDs), and any immune and/orinflammatory modulators. Thus, compounds of this invention may beadministered with:

-   -   Modulators of other members of the TNF/TNF receptor family,        including TNF antagonists, such as etanercept (Enbrel™),        sTNF-RI, onercept, D2E7, and Remicade™.    -   Nerve growth factor (NGF) modulators.    -   IL-1 inhibitors, including IL-1ra molecules such as anakinra and        more recently discovered IL-1ra-like molecules such as IL-1Hy1        and IL-1Hy2; IL-1 “trap” molecules as described in U.S. Pat. No.        5,844,099, issued Dec. 1, 1998; IL-1 antibodies; solubilized        IL-1 receptor, and the like.    -   IL-6 inhibitors (e.g., antibodies to IL-6).    -   IL-8 inhibitors (e.g., antibodies to IL-8).    -   IL-18 inhibitors (e.g., IL-18 binding protein, solubilized IL-18        receptor, or IL-18 antibodies).    -   Interleukin-1 converting enzyme (ICE) modulators.    -   insulin-like growth factors (IGF-1, IGF-2) and modulators        thereof.    -   Transforming growth factor-β (TGF-β), TGF-β family members, and        TGF-β modulators.    -   Fibroblast growth factors FGF-1 to FGF-10, and FGF modulators.    -   Osteoprotegerin (OPG), OPG analogues, osteoprotective agents,        and antibodies to OPG-ligand (OPG-L).    -   bone anabolic agents, such as parathyroid hormone (PTH), PTH        fragments, and molecules incorporating PTH fragments (e.g., PTH        (1-34)-Fc).    -   PAF antagonists.    -   Keratinocyte growth factor (KGF), KGF-related molecules (e.g.,        KGF-2), and KGF modulators.    -   COX-2 inhibitors, such as Celebrex™ and Vioxx™    -   Prostaglandin analogs (e.g., E series prostaglandins).    -   Matrix metalloproteinase (MMP) modulators.    -   Nitric oxide synthase (NOS) modulators, including modulators of        inducible NOS.    -   Modulators of glucocorticoid receptor.    -   Modulators of glutamate receptor.    -   Modulators of lipopolysaccharide (LPS) levels.    -   Anti-cancer agents, including inhibitors of oncogenes (e.g.,        fos, jun) and interferon.    -   Noradrenaline and modulators and mimetics thereof.

Pharmaceutical Compositions

In General.

The present invention also provides methods of using pharmaceuticalcompositions of the inventive compounds. Such pharmaceuticalcompositions may be for administration for injection, or for oral,pulmonary, nasal, transdermal or other forms of administration. Ingeneral, the invention encompasses pharmaceutical compositionscomprising effective amounts of a compound of the invention togetherwith pharmaceutically acceptable diluents, preservatives, solubilizers,emulsifiers, adjuvants and/or carriers. Such compositions includediluents of various buffer content (e.g., Tris-HCl, acetate, phosphate),pH and ionic strength; additives such as detergents and solubilizingagents (e.g., Tween 80, Polysorbate 80), anti-oxidants (e.g., ascorbicacid, sodium metabisulfite), preservatives (e.g., Thimersol, benzylalcohol) and bulking substances (e.g., lactose, mannitol); incorporationof the material into particulate preparations of polymeric compoundssuch as polylactic acid, polyglycolic acid, etc. or into liposomes.Hyaluronic acid may also be used, and this may have the effect ofpromoting sustained duration in the circulation. Such compositions mayinfluence the physical state, stability, rate of in vivo release, andrate of in vivo clearance of the present proteins and derivatives. See,e.g., Remington's Pharmaceutical Sciences, 18th Ed. (1990, MackPublishing Co., Easton, Pa. 18042) pages 1435-1712 which are hereinincorporated by reference in their entirety. The compositions may beprepared in liquid form, or may be in dried powder, such as lyophilizedform. Implantable sustained release formulations are also contemplated,as are transdermal formulations.

Oral Dosage Forms.

Contemplated for use herein are oral solid dosage forms, which aredescribed generally in Chapter 89 of Remington's Pharmaceutical Sciences(1990), 18th Ed., Mack Publishing Co. Easton Pa. 18042, which is hereinincorporated by reference in its entirety. Solid dosage forms includetablets, capsules, pills, troches or lozenges, cachets or pellets. Also,liposomal or proteinoid encapsulation may be used to formulate thepresent compositions (as, for example, proteinoid microspheres reportedin U.S. Pat. No. 4,925,673). Liposomal encapsulation may be used and theliposomes may be derivatized with various polymers (e.g., U.S. Pat. No.5,013,556). A description of possible solid dosage forms for thetherapeutic is given in Chapter 10 of Marshall, K., Modern Pharmaceutics(1979), edited by G. S. Banker and C. T. Rhodes, herein incorporated byreference in its entirety. In general, the formulation will include theinventive compound, and inert ingredients which allow for protectionagainst the stomach environment, and release of the biologically activematerial in the intestine.

Also specifically contemplated are oral dosage forms of the aboveinventive compounds. If necessary, the compounds may be chemicallymodified so that oral delivery is efficacious. Generally, the chemicalmodification contemplated is the attachment of at least one moiety tothe compound molecule itself, where said moiety permits (a) inhibitionof proteolysis; and (b) uptake into the blood stream from the stomach orintestine. Also desired is the increase in overall stability of thecompound and increase in circulation time in the body. Moieties usefulas covalently attached vehicles in this invention may also be used forthis purpose. Examples of such moieties include: PEG, copolymers ofethylene glycol and propylene glycol, carboxymethyl cellulose, dextran,polyvinyl alcohol, polyvinyl pyrrolidone and polyproline. See, forexample, Abuchowski and Davis, Soluble Polymer-Enzyme Adducts, Enzymesas Drugs (1981), Hocenberg and Roberts, eds., Wiley-Interscience, NewYork, N.Y., pp. 367-83; Newmark, et al. (1982), I. Appl. Biochem.4:185-9. Other polymers that could be used are poly-1,3-dioxolane andpoly-1,3,6-tioxocane. Preferred for pharmaceutical usage, as indicatedabove, are PEG moieties.

For oral delivery dosage forms, it is also possible to use a salt of amodified aliphatic amino acid, such as sodium N-(8-[2-hydroxybenzoyl]amino) caprylate (SNAC), as a carrier to enhance absorption of thetherapeutic compounds of this invention. The clinical efficacy of aheparin formulation using SNAC has been demonstrated in a Phase II trialconducted by Emisphere Technologies. See U.S. Pat. No. 5,792,451, “Oraldrug delivery composition and methods”.

The compounds of this invention can be included in the formulation asfine multiparticulates in the form of granules or pellets of particlesize about 1 mm. The formulation of the material for capsuleadministration could also be as a powder, lightly compressed plugs oreven as tablets. The therapeutic could be prepared by compression.

Colorants and flavoring agents may all be included. For example, theprotein (or derivative) may be formulated (such as by liposome ormicrosphere encapsulation) and then further contained within an edibleproduct, such as a refrigerated beverage containing colorants andflavoring agents.

One may dilute or increase the volume of the compound of the inventionwith an inert material. These diluents could include carbohydrates,especially mannitol, α-lactose, anhydrous lactose, cellulose, sucrose,modified dextrans and starch. Certain inorganic salts may also be usedas fillers including calcium triphosphate, magnesium carbonate andsodium chloride. Some commercially available diluents are Fast-Flo,Emdex, STA-Rx 1500, Emcompress and Avicell.

Disintegrants may be included in the formulation of the therapeutic intoa solid dosage form. Materials used as disintegrants include but are notlimited to starch including the commercial disintegrant based on starch,Explotab. Sodium starch glycolate, Amberlite, sodiumcarboxymethylcellulose, ultramylopectin, sodium alginate, gelatin,orange peel, acid carboxymethyl cellulose, natural sponge and bentonitemay all be used. Another form of the disintegrants are the insolublecationic exchange resins. Powdered gums may be used as disintegrants andas binders and these can include powdered gums such as agar, Karaya ortragacanth. Alginic acid and its sodium salt are also useful asdisintegrants.

Binders may be used to hold the therapeutic agent together to form ahard tablet and include materials from natural products such as acacia,tragacanth, starch and gelatin. Others include methyl cellulose (MC),ethyl cellulose (EC) and carboxymethyl cellulose (CMC). Polyvinylpyrrolidone (PVP) and hydroxypropylmethyl cellulose (HPMC) could both beused in alcoholic solutions to granulate the therapeutic.

An antifrictional agent may be included in the formulation of thetherapeutic to prevent sticking during the formulation process.Lubricants may be used as a layer between the therapeutic and the diewall, and these can include but are not limited to; stearic acidincluding its magnesium and calcium salts, polytetrafluoroethylene(PTFE), liquid paraffin, vegetable oils and waxes. Soluble lubricantsmay also be used such as sodium lauryl sulfate, magnesium laurylsulfate, polyethylene glycol of various molecular weights, Carbowax 4000and 6000.

Glidants that might improve the flow properties of the drug duringformulation and to aid rearrangement during compression might be added.The glidants may include starch, talc, pyrogenic silica and hydratedsilicoaluminate.

To aid dissolution of the compound of this invention into the aqueousenvironment a surfactant might be added as a wetting agent. Surfactantsmay include anionic detergents such as sodium lauryl sulfate, dioctylsodium sulfosuccinate and dioctyl sodium sulfonate. Cationic detergentsmight be used and could include benzalkonium chloride or benzethoniumchloride. The list of potential nonionic detergents that could beincluded in the formulation as surfactants are lauromacrogol 400,polyoxyl 40 stearate, polyoxyethylene hydrogenated castor oil 10, 50 and60, glycerol monostearate, polysorbate 40, 60, 65 and 80, sucrose fattyacid ester, methyl cellulose and carboxymethyl cellulose. Thesesurfactants could be present in the formulation of the protein orderivative either alone or as a mixture in different ratios.

Additives may also be included in the formulation to enhance uptake ofthe compound. Additives potentially having this property are forinstance the fatty acids oleic acid, linoleic acid and linolenic acid.

Controlled release formulation may be desirable. The compound of thisinvention could be incorporated into an inert matrix which permitsrelease by either diffusion or leaching mechanisms; e.g., gums. Slowlydegenerating matrices may also be incorporated into the formulation,e.g., alginates, polysaccharides. Another form of a controlled releaseof the compounds of this invention is by a method based on the Orostherapeutic system (Alza Corp.), i.e., the drug is enclosed in asemipermeable membrane which allows water to enter and push drug outthrough a single small opening due to osmotic effects. Some entericcoatings also have a delayed release effect.

Other coatings may be used for the formulation. These include a varietyof sugars which could be applied in a coating pan. The therapeutic agentcould also be given in a film coated tablet and the materials used inthis instance are divided into 2 groups. The first are the nonentericmaterials and include methyl cellulose, ethyl cellulose, hydroxyethylcellulose, methylhydroxy-ethyl cellulose, hydroxypropyl cellulose,hydroxypropyl-methyl cellulose, sodium carboxy-methyl cellulose,providone and the polyethylene glycols. The second group consists of theenteric materials that are commonly esters of phthalic acid. A mix ofmaterials might be used to provide the optimum film coating. Filmcoating may be carried out in a pan coater or in a fluidized bed or bycompression coating.

Pulmonary Delivery Forms.

Also contemplated herein is pulmonary delivery of the present protein(or derivatives thereof). The protein (or derivative) is delivered tothe lungs of a mammal while inhaling and traverses across the lungepithelial lining to the blood stream. (Other reports of this includeAdjei et al., Pharma. Res. (1990) 7: 565-9; Adjei et al. (1990),Internatl. J. Pharmaceutics 63: 135-44 (leuprolide acetate); Braquet etal. (1989), J. Cardiovasc. Pharmacol. 13 (suppl. 5): s. 143-146(endothelin-1); Hubbard et al. (1989), Annals Int. Med. 3: 206-12(α1-antitrypsin); Smith et al. (1989), J. Gin. Invest. 84: 1145-6(α1-proteinase); Oswein et al. (March 1990), “Aerosolization ofProteins”, Proc. Symp. Resp. Drug Delivery II, Keystone, Colo.(recombinant human growth hormone); Debs et al. (1988), I. Immunol. 140:3482-8 (interferon-γ and tumor necrosis factor α) and Platz et al., U.S.Pat. No. 5,284,656 (granulocyte colony stimulating factor).

Contemplated for use in the practice of this invention are a wide rangeof mechanical devices designed for pulmonary delivery of therapeuticproducts, including but not limited to nebulizers, metered doseinhalers, and powder inhalers, all of which are familiar to thoseskilled in the art. Some specific examples of commercially availabledevices suitable for the practice of this invention are the Ultraventnebulizer, manufactured by Mallinckrodt, Inc., St. Louis, Mo.; the AcornII nebulizer, manufactured by Marquest Medical Products, Englewood,Colo.; the Ventolin metered dose inhaler, manufactured by Glaxo Inc.,Research Triangle Park, N.C.; and the Spinhaler powder inhaler,manufactured by Fisons Corp., Bedford, Mass.

All such devices require the use of formulations suitable for thedispensing of the inventive compound. Typically, each formulation isspecific to the type of device employed and may involve the use of anappropriate propellant material, in addition to diluents, adjuvantsand/or carriers useful in therapy.

The inventive compound should most advantageously be prepared inparticulate form with an average particle size of less than 10 μm (ormicrons), most preferably 0.5 to 5 μm, for most effective delivery tothe distal lung.

Pharmaceutically acceptable carriers include carbohydrates such astrehalose, mannitol, xylitol, sucrose, lactose, and sorbitol. Otheringredients for use in formulations may include DPPC, DOPE, DSPC andDOPC. Natural or synthetic surfactants may be used. PEG may be used(even apart from its use in derivatizing the protein or analog).Dextrans, such as cyclodextran, may be used. Bile salts and otherrelated enhancers may be used. Cellulose and cellulose derivatives maybe used. Amino acids may be used, such as use in a buffer formulation.

Also, the use of liposomes, microcapsules or microspheres, inclusioncomplexes, or other types of carriers is contemplated.

Formulations suitable for use with a nebulizer, either jet orultrasonic, will typically comprise the inventive compound dissolved inwater at a concentration of about 0.1 to 25 mg of biologically activeprotein per mL of solution. The formulation may also include a bufferand a simple sugar (e.g., for protein stabilization and regulation ofosmotic pressure). The nebulizer formulation may also contain asurfactant, to reduce or prevent surface induced aggregation of theprotein caused by atomization of the solution in forming the aerosol.

Formulations for use with a metered-dose inhaler device will generallycomprise a finely divided powder containing the inventive compoundsuspended in a propellant with the aid of a surfactant. The propellantmay be any conventional material employed for this purpose, such as achlorofluorocarbon, a hydrochlorofluorocarbon, a hydrofluorocarbon, or ahydrocarbon, including trichlorofluoromethane, dichlorodifluoromethane,dichlorotetrafluoroethanol, and 1,1,1,2- tetrafluoroethane, orcombinations thereof. Suitable surfactants include sorbitan trioleateand soya lecithin. Oleic acid may also be useful as a surfactant.

Formulations for dispensing from a powder inhaler device will comprise afinely divided dry powder containing the inventive compound and may alsoinclude a bulking agent, such as lactose, sorbitol, sucrose, mannitol,trehalose, or xylitol in amounts which facilitate dispersal of thepowder from the device, e.g., 50 to 90% by weight of the formulation.

Nasal Delivery Forms.

Nasal delivery of the inventive compound is also contemplated. Nasaldelivery allows the passage of the protein to the blood stream directlyafter administering the therapeutic product to the nose, without thenecessity for deposition of the product in the lung. Formulations fornasal delivery include those with dextran or cyclodextran. Delivery viatransport across other mucous membranes is also contemplated.

Dosages.

The dosage regimen involved in a method for treating the above-describedconditions will be determined by the attending physician, consideringvarious factors which modify the action of drugs, e.g. the age,condition, body weight, sex and diet of the patient, the severity of anyinfection, time of administration and other clinical factors. Generally,the daily regimen should be in the range of 0.1-1000 micrograms of theinventive compound per kilogram of body weight, preferably 0.1-150micrograms per kilogram.

Specific Preferred Embodiments

The inventors have determined preferred structures for the preferredpeptides listed in Table 4 below. The symbol “A” may be any of thelinkers described herein or may simply represent a normal peptide bond(i.e., so that no linker is present). Tandem repeats and linkers areshown separated by dashes for clarity.

TABEL 4  Preferred embodiments SEQ ID Sequence/structure NO:LPGCKWDLLIKQWVCDPL-Λ-V¹ 44 V¹-Λ-LPGCKWDLLIKQWVCDPL 45LPGCKWDLLIKQWVCDPL-Λ- 46 LPGCKWDLLIKQWVCDPL-Λ-V¹V¹-Λ-LPGCKWDLLIKQWVCDPL-Λ- 47 LPGCKWDLLIKQWVCDPL SADCYFDILTKSDVCTSS-Λ-V¹48 V¹-Λ-SADCYFDILTKSDVCTSS 49 SADCYFDILTKSDVTSS-Λ-SADCYF 50DILTKSDVTSS-Λ-V¹ V¹-Λ-SADCYFDILTKSDVTSS-Λ- 51 SADCYFDILTKSDVTSSFHDCKWDLLTKQWVCHGL-Λ-V¹ 52 V¹-Λ-FHDCKWDLLTKQWVCHGL 53FHDCKWDLLTKQWVCHGL-Λ- 54 FHDCKWDLLTKQWVCHGL-Λ-V¹V¹-Λ-FHDCKWDLLTKQWVCHGL-Λ- 55 FHDCKWDLLTKQWVCHGL“V¹” is an Fc domain as defined previously herein. In addition to thoselisted in Table 4, the inventors further contemplate heterodimers inwhich each strand of an Fc dimer is linked to a different peptidesequence; for example, wherein each Fc is linked to a different sequenceselected from Table 2.

All of the compounds of this invention can be prepared by methodsdescribed in PCT appl. no. WO 99/25044.

The invention will now be further described by the following workingexamples, which are illustrative rather than limiting.

Example 1 Peptides Peptide Phage Display

1. Magnetic Bead Preparation

A. Fc-TALL-1 Immobilization on Magnetic Beads

The recombinant Fc-TALL-1 protein was immobilized on the Protein ADynabeads (Dynal) at a concentration of 8 μg of Fc-TALL-1 per 100 μl ofthe bead stock from the manufacturer. By drawing the beads to one sideof a tube using a magnet and pipetting away the liquid, the beads werewashed twice with the phosphate buffer saline (PBS) and resuspended inPBS. The Fc-TALL-1 protein was added to the washed beads at the aboveconcentration and incubated with rotation for 1 hour at roomtemperature. The Fc-TALL-1 coated beads were then blocked by addingbovine serum albumin (BSA) to 1% final concentration and incubatingovernight at 4° C. with rotation. The resulting Fe-TALL-1 coated beadswere then washed twice with PB ST (PBS with 0.05% Tween-20) before beingsubjected to the selection procedures.

B. Negative Selection Bead Preparation

Additional beads were also prepared for negative selections. For eachpanning condition, 250 μl of the bead stock from the manufacturer wassubjected to the above procedure (section 1A) except that the incubationstep with Fc-TALL-1 was omitted. In the last washing step, the beadswere divided into five 50 μl aliquots.

2. Selection of TALL-1 Binding Phage

A. Overall Strategy

Two filamentous phage libraries, TN8-IX (5×10⁹ independenttransformants) and TN12-I (1.4×10⁹ independent transformants) (DyaxCorp.), were used to select for TALL-1 binding phage. Each library wassubjected to either pH 2 elution or ‘bead elution’ (section 2E).Therefore, four different panning conditions were carried out for theTALL-1 project (TN8-IX using the pH2 elution method, TN8-IX using thebead elution method, TN12-I the using p112 elution method, and TN 12-Iusing the bead elution method). Three rounds of selection were performedfor each condition.

B. Negative Selection

For each panning condition, about 100 random library equivalent (5×10¹¹pfu for TN8-IX and 1.4×10¹¹ pfu for TN12-I) was aliquoted from thelibrary stock and diluted to 300 μl with PB ST. After the last washingliquid was drawn out from the first 50 μl aliquot of the beads preparedfor negative selections (section 1B), the 300 μl diluted library stockwas added to the beads. The resulting mixture was incubated for 10minutes at room temperature with rotation. The phage supernatant wasdrawn out using the magnet and added to the second 50 μl aliquot foranother negative selection step. In this way, five negative selectionsteps were performed.

C. Selection Using the Fc-TALL-1 Protein Coated Beads

The phage supernatant after the last negative selection step (section1B) was added to the Fc-TALL-1 coated beads after the last washing step(section 1A). This mixture was incubated with rotation for two hours atroom temperature, allowing specific phage to bind to the target protein.After the supernatant is discarded, the beads were washed seven timeswith PBST.

D. pH2 Elution of Bound Phage

After the last washing step (section 2C), the bound phages were elutedfrom the magnetic beads by adding 200 μl of CBST (50 mM sodium citrate,150 mM sodium chloride, 0.05% Tween-20, pH2). After 5 minute incubationat room temperature, the liquid containing the eluted phage were drawnout and transferred to another tube. The elution step was repeated againby adding 200 μl of CBST and incubating for 5 minutes. The liquids fromtwo elution steps were added together, and 100 μl of 2 M Tris solution(pH 8) was added to neutralize the pH. 500 μl of Min A Salts solution(60 mM K₂HPO₄, 33 mM KH₂PO₄, 7.6 mM (NH₄)SO₄, and 1.7 mM sodium citrate)was added to make the final volume to 1 ml.

E. ‘Bead Elution’

After the final washing liquid was drawn out (section 2C), 1 ml of Min Asalts solution was added to the beads. This bead mixture was addeddirectly to a concentrated bacteria sample for infection (section 3A and3B).

3. Amplification

A. Preparation of Plating Cells

Fresh E. Coli. (XL-1 Blue MRF′) culture was grown to OD₆₀₀=0.5 in LBmedia containing 12.5 μg/ml tetracycline. For each panning condition, 20ml of this culture was chilled on ice and centrifuged. The bacteriapellet was resuspended in 1 ml of the Min A Salts solution.

B. Transduction

Each mixture from different elution methods (section 2D and 2E) wasadded to a concentrated bacteria sample (section 3A) and incubated at37° C. for 15 minutes. 2 ml of NZCYM media (2λNZCYM, 50 μg/mlampicillin) was added to each mixture and incubated at room temperaturefor 15 minutes. The resulting 4 ml solution was plated on a large NZCYMagar plate containing 50 μg/ml ampicillin and incubated overnight at 37°C.

C. Phage Harvesting

Each of the bacteria/phage mixture that was grown overnight on a largeNZCYM agar plate (section 3B) was scraped off in 35 ml of LB media, andthe agar plate was further rinsed with additional 35 ml of LB media. Theresulting bacteria/phage mixture in LB media was centrifuged to pelletthe bacteria away. 50 ml the of the phage supernatant was transferred toa fresh tube, and 12.5 ml of PEG solution (20% PEG8000, 3.5M ammoniumacetate) was added and incubated on ice for 2 hours to precipitatephages. Precipitated phages were centrifuged down and resuspended in 6ml of the phage resuspension buffer (250 mM NaCl, 100 mM Tris pH8, 1 mMEDTA). This phage solution was further purified by centrifuging away theremaining bacteria and precipitating the phage for the second time byadding 1.5 ml of the PEG solution. After a centrifugation step, thephage pellet was resuspended in 400 IA of PBS. This solution wassubjected to a final centrifugation to rid of remaining bacteria debris.The resulting phage preparation was titered by a standard plaqueformation assay (Molecular Cloning, Maniatis et al 3^(rd) Edition).

4. Two More Rounds of Selection and Amplification.

In the second round, the amplified phage (10¹⁰ pfu) from the first round(section 3C) was used as the input phage to perform the selection andamplification steps (sections 2 and 3). The amplified phage (10¹⁰ pfu)from the 2^(nd) round in turn was used as the input phage to perform3^(rd) round of selection and amplification (sections 2 and 3). Afterthe elution steps (sections 2D and 2E) of the 3^(rd) round, a smallfraction of the eluted phage was plated out as in the plaque formationassay (section 3C). Individual plaques were picked and placed into 96well microtiter plates containing 100 μl of TE buffer in each well.These master plates were incubated in a 37° C. incubator for 1 hour toallow phages to elute into the TE buffer.

5. Clonal Analysis (Phage ELISA and Sequencing)

The phage clones were analyzed by phage ELISA and sequencing methods.The sequences were ranked based on the combined results from these twoassays.

A. Phage ELISA

An XL-1 Blue MRF′ culture was grown until OD₆₀₀, reaches 0.5. 30 μl ofthis culture was aliquoted into each well of a 96 well microtiter plate.10 μl of eluted phage (section 4) was added to each well and allowed toinfect bacteria for 15 min at room temperature. 130 μl of LB mediacontaining 12.5 μl of tetracycline and 50 μg/ml of ampicillin was addedto each well. The microtiter plate was then incubated overnight at 37°C. The recombinant TALL-1 protein (1 μg/ml in PBS) was allowed to coatonto the 96-well Maxisorp plates (NUNC) overnight and 4° C. As acontrol, the recombinant Fc-Trail protein was coated onto a separateMaxisorp plate at the same molar concentration as the TALL-1 protein.

On the following day, liquids in the protein coated Maxisorp plates werediscarded, and each well was blocked with 300 μl of 2% BSA solution at37° C. for one hour. The BSA solution was discarded, and the wells werewashed three times with the PBST solution. After the last washing step,50 μl of PBST was added to each well of the protein coated Maxisorpplates. Each of the 50 μl overnight cultures in the 96 well microtiterplate was transferred to the corresponding wells of the TALL-1 coatedplates as well as the control Fc-Trail coated plates. The 100 μlmixtures in the two kinds of plates were incubated for 1 hour at roomtemperature. The liquid was discarded from the Maxisorp plates, and thewells were washed five times with PBST. The HRP-conjugated anti-M13antibody (Pharmacia) was diluted to 1:7,500, and 100 μl of the dilutedsolution was added to each well of the Maxisorp plates for 1 hourincubation at room temperature. The liquid was again discarded and thewells were washed seven times with PBST. 100 μl of tetramethylbenzidine(TMB) substrate (Sigma) was added to each well for the color reaction todevelop, and the reaction was stopped with 50 μl of the 5 N H₂SO₄solution. The OD₄₅₀ was read on a plate reader (Molecular Devices).

B. Sequencing of the Phage Clones.

For each phage clone, the sequencing template was prepared by a PCRmethod. The following oligonucleotide pair was used to amplify about 500nucleotide fragment:

primer #1 (SEQ ID NO: 56) (5′-CGGCGCAACTATCGGTATCAAGCTG-3′) and primer #2 (SEQ ID NO: 57) (5′-CATGTACCGTAACACTGAGTTTCGTC-3′).The following mixture was prepared for each clone.

Reagents volume (μL)/tube dH₂0 26.25 50% glycerol 10 10B PCR Buffer (w/oMgCl₂) 5 25 mM MgCl₂ 4 10 mM dNTP mix 1 100 μM primer 1 0.25 100 μMprimer 2 0.25 Taq polymerase 0.25 Phage in TE (section 4) 3 Finalreaction volume 50

The thermocycler (GeneAmp PCR System 9700, Applied Biosystems) was usedto run the following program: 94° C. for 5 min; [94° C. for 30 sec, 55°C. for 30 sec, 72° C. for 45 sec.]×30 cycles; 72° C. for 7 min; cool to4° C. The PCR product was checked by running 5 μl of each PCR reactionon a 1% agarose gel. The PCR product in the remaining 45 μl from eachreaction was cleaned up using the QlAquick Multiwell PCR Purificationkit (Qiagen), following the manufacturer's protocol. The resultingproduct was then sequenced using the ABI 377 Sequencer (Perkin-Elmer)following the manufacturer recommended protocol.

6. Sequence Ranking and Consensus Sequence Determination

A. Sequence Ranking

The peptide sequences that were translated from variable nucleotidesequences (section 5B) were correlated to ELISA data. The clones thatshowed high OD₄₅₀ in the TALL-1 coated wells and low OD₄₅₀ in theFc-Trail coated wells were considered more important. The sequences thatoccur multiple times were also considered important. Candidate sequenceswere chosen based on these criteria for further analysis as peptides orpeptibodies. Five and nine candidate peptide sequences were selectedfrom the TN8-IX and TN12-I libraries, respectively.

B. Consensus Sequence Determination

The majority of sequences selected from the TN12-I library contained avery conserved DBL motif. This motif was also observed in sequencesselected from the TN8-IB library as well. Another motif, PFPWE (SEQ IDNO: 110) was also observed in sequences obtained from the TN8-IBlibrary.

A consensus peptide, FHDCKWDLLTKQWVCHGL (SEQ ID NO: 58), was designedbased on the DBL motif. Since peptides derived from the TN12-I librarywere the most active ones, the top 26 peptide sequences based on theabove ranking criteria (section 5A) were aligned by the DBL motif. Theunderlined “core amino acid sequence” was obtained by determining theamino acid that occur the most in each position. The two cysteinesadjacent to the core sequences were fixed amino acids in the TN12-Ilibrary. The rest of the amino acid sequence in the consensus peptide istaken from one of the candidate peptides, TALL-1-12-10 (Table 2, SEQ IDNO: 37). The peptide and peptibody that was derived from this consensussequence were most active in the B cell proliferation assay.

Example 2 Peptibodies

A set of 12 TALL-1 inhibitory peptibodies (Table 5) was constructed inwhich a monomer of each peptide was fused in-frame to the Fc region ofhuman IgG1. Each TALL-1 inhibitory peptibody was constructed byannealing the pairs of oligonucleotides shown in Table 6 to generate aduplex encoding the peptide and a linker comprised of 5 glycine residuesand one valine residue as an NdeI to SalI fragment. These duplexmolecules were ligated into a vector (pAMG21-RANK-Fc, described herein)containing the human Fc gene, also digested with {right arrow over(Nde)}I and {right arrow over (Sal)}I. The resulting ligation mixtureswere transformed by electroporation into E. coli strain 2596 cells(GM221, described herein). Clones were screened for the ability toproduce the recombinant protein product and to possess the gene fusionhaving the correct nucleotide sequence. A single such clone was selectedfor each of the peptibodies. The nucleotide and amino acid sequences ofthe fusion proteins are shown in FIG. 4A through 4F.

TABLE 5  Peptide sequences and oligonucleotides used togenerate TALL-1 inhibitory peptibodies. Pepti- Sense Antisense bodyoligo- oligo- SEQ ID Peptide  nucleo- nucleo- Peptibody NO Sequence tidetide TALL-1-8-1-a 29 PGTCFPFPWEC 2517-24 2517-25 THA TALL-1-8-2-a 30WGACWPFPWEC 2517-26 2517-27 FKE TALL-1-8-4-a 31 VPFCDLLTKHC 2517-282517-29 FEA TALL-1-12-4- 32 GSRCKYKWDVL 2517-30 2517-31 a TKQCFHHTALL-1-12-3- 33 LPGCKWDLLIK 2517-32 2517-33 a QWVCDPL TALL-1-12-5- 34SADCYFDILTK 2517-34 2517-35 a SDVCTSS TALL-1-12-8- 35 SDDCMYDQLTR2517-36 2517-37 a MFICSNL TALL-1-12-9- 36 DLNCKYDELTY 2521-92 2521-93 aKEWCQFN TALL-1-12- 37 FHDCKYDLLTR 2521-94 2521-95 10-a QMVCHGLTALL-1-12- 38 RNHCFWDHLLK 2521-96 2521-97 11-a QDICPSP TALL-1-12- 39ANQCWWDSLTK 2521-98 2521-99 14-a KNVCEFF TALL-1- 58 FHDCKWDLLTK 2551-482551-49 consensus QWVCHGL

TABLE 5B  TALL-1 inhibitory peptibodies. Peptibody SEQ ID Peptibody NOPeptide Sequence TALL-I-8-1-a 111MPGTCFPFPW ECTHAGGGGG VDKTHTCPPC PAPELLGGPSVFLFPPKPKD TLMISRTPEV TCVVVDVSHE DPEVKFNWYVDGVEVHNAKT KPREEQYNST YRVVSVLTVL HQDWLNGKEYKCKVSNKALP APIEKTISKA KGQPREPQVY TLPPSRDELTKNQVSLTCLV KGFYPSDIAV EWESNGQPEN NYKTTPPVLDSDGSFFLYSK LTVDKSRWQQ GNVFSCSVMH EALHNHYTQK SLSLSPGK TALL-1-8-2-a 112MWGACWPFPW ECFKEGGGGG VDKTHTCPPC PAPELLGGPSVFLFPPKPKD TLMISRTPEV TCVVVDVSHE DPEVKFNWYVDGVEVHNAKT KPREEQYNST YRVVSVLTVL HQDWLNGKEYKCKVSNKALP APIEKTISKA KGQPREPQVY TLPPSRDELTKNQVSLTCLV KGFYPSDIAV EWESNGQPEN NYKTTPPVLDSDGSFFLYSK LTVDKSRWQQ GNVFSCSVMH EALHNHYTQK SLSLSPGK TALL-1-8-4-a 113MVPFCDLLTK HCFEAGGGGG VDKTHTCPPC PAPELLGGPSVFLFPPKPKD TLMISRTPEV TCVVVDVSHE DPEVKFNWYVDGVEVHNAKT KPREEQYNST YRVVSVLTVL HQDWLNGKEYKCKVSNKALP APIEKTISKA KGQPREPQVY TLPPSRDELTKNQVSLTCLV KGFYPSDIAV EWESNGQPEN NYKTTPPVLDSDGSFFLYSK LTVDKSRWQQ GNVFSCSVMH EALHNHYTQK SLSLSPGK TALL-1-12-4-a 114MGSRCKYKWD VLTKQCFHHG GGGGVDKTHT CPPCPAPELLGGPSVFLFPP KPKDTLMISR TPEVTCVVVD VSHEDPEVKFNWYVDGVEVH NAKTKPREEQ YNSTYRVVSV LTVLHQDWLNGKEYKCKVSN KALPAPIEKT ISKAKGQPRE PQVYTLPPSRDELTKNQVSL TCLVKGFYPS DIAVEWESNG QPENNYKTTPPVLDSDGSFF LYSKLTVDKS RWQQGNVFSC SVMHEALHNH YTQKSLSLSP GK TALL-1-12-3-a115 MLPGCKWDLL IKQWVCDPLG GGGGVDKTHT CPPCPAPELLGGPSVFLFPP KPKDTLMISR TPEVTCVVVD VSHEDPEVKFNWYVDGVEVH NAKTKPREEQ YNSTYRVVSV LTVLHQDWLNGKEYKCKVSN KALPAPIEKT ISKAKGQPRE PQVYTLPPSRDELTKNQVSL TCLVKGFYPS DIAVEWESNG QPENNYKTTPPVLDSDGSFF LYSKLTVDKS RWQQGNVFSC SVMHEALHNH YTQKSLSLSP GK TALL-1-12-5-a116 MSADCYFDIL TKSDVCTSSG GGGG VDKTHT CPPCPAPELLGGPSVFLFPP KPKDTLMISR TPEVTCVVVD VSHEDPEVKFNWYVDGVEVH NAKTKPREEQ YNSTYRVVSV LTVLHQDWLNGKEYKCKVSN KALPAPIEKT ISKAKGQPRE PQVYTLPPSRDELTKNQVSL TCLVKGFYPS DIAVEWESNG QPENNYKTTPPVLDSDGSFF LYSKLTVDKS RWQQGNVFSC SVMHEALHNH YTQKSLSLSP GK TALL-1-12-8-a117 MSDDCMYDQL TRMFICSNLG GGGGVDKTHT CPPCPAPELLGGPSVFLFPP KPKDTLMISR TPEVTCVVVD VSHEDPEVKFNWYVDGVEVH NAKTKPREEQ YNSTYRVVSV LTVLHQDWLNGKEYKCKVSN KALPAPIEKT ISKAKGQPRE PQVYTLPPSRDELTKNQVSL TCLVKGFYPS DIAVEWESNG QPENNYKTTPPVLDSDGSFF LYSKLTVDKS RWQQGNVFSC SVMHEALHNH YTQKSLSLSP GK TALL-1-12-9-a118 MDLNCKYDEL TYKEWCQFNG GGGGVDKTHT CPPCPAPELLGGPSVFLFPP KPKDTLMISR TPEVTCVVVD VSHEDPEVKFNWYVDGVEVH NAKTKPREEQ YNSTYRVVSV LTVLHQDWLNGKEYKCKVSN KALPAPIEKT ISKAKGQPRE PQVYTLPPSRDELTKNQVSL TCLVKGFYPS DIAVEWESNG QPENNYKTTPPVLDSDGSFF LYSKLTVDKS RWQQGNVFSC SVMHEALHNH YTQKSLSLSP GK TALL-1-12-10-a119 MFHDCKYDLL TRQMVCHGLG GGGGVDKTHT CPPCPAPELLGGPSVFLFPP KPKDTLMISR TPEVTCVVVD VSHEDPEVKFNWYVDGVEVH NAKTKPREEQ YNSTYRVVSV LTVLHQDWLNGKEYKCKVSN KALPAPIEKT ISKAKGQPRE PQVYTLPPSRDELTKNQVSL TCLVKGFYPS DIAVEWESNG QPENNYKTTPPVLDSDGSFF LYSKLTVDKS RWQQGNVFSC SVMHEALHNH YTQKSLSLSP GK TALL-1-12-11-a120 MRNHCFWDHL LKQDICPSPG GGGGVDKTHT CPPCPAPELLGGPSVFLFPP KPKDTLMISR TPEVTCVVVD VSHEDPEVKFNWYVDGVEVH NAKTKPREEQ YNSTYRVVSV LTVLHQDWLNGKEYKCKVSN KALPAPIEKT ISKAKGQPRE PQVYTLPPSRDELTKNQVSL TCLVKGFYPS DIAVEWESNG QPENNYKTTPPVLDSDGSFF LYSKLTVDKS RWQQGNVFSC SVMHEALHNH YTQKSLSLSP GK TALL-1-12-14-a121 MANQCWWDSL TKKNVCEFFG GGGGVDKTHT CPPCPAPELLGGPSVFLFPP KPKDTLMISR TPEVTCVVVD VSHEDPEVKFNWYVDGVEVH NAKTKPREEQ YNSTYRVVSV LTVLHQDWLNGKEYKCKVSN KALPAPIEKT ISKAKGQPRE PQVYTLPPSRDELTKNQVSL TCLVKGFYPS DIAVEWESNG QPENNYKTTPPVLDSDGSFF LYSKLTVDKS RWQQGNVFSC SVMHEALHNH YTQKSLSLSP GK TALL-1- 122MFHDCKWDLL TKQWVCHGLG GGGGVDKTHT CPPCPAPELL consensusGGPSVFLFPP KPKDTLMISR TPEVTCVVVD VSHEDPEVKFNWYVDGVEVH NAKTKPREEQ YNSTYRVVSV LTVLHQDWLNGKEYKCKVSN KALPAPIEKT ISKAKGQPRE PQVYTLPPSRDELTKNQVSL TCLVKGFYPS DIAVEWESNG QPENNYKTTPPVLDSDGSFF LYSKLTVDKS RWQQGNVFSC SVMHEALHNH YTQKSLSLSP GK TALL-112-3 123 MLPGCKWDLL IKQWVCDPLG SGSATGGSGS TASSGSGSAT tandemHMLPGCKWDL LIKQWVCDPL GGGGGVDKTH TCPPCPAPEL dimerLGGPSVFLFP PKPKDTLMIS RTPEVTCVVV DVSHEDPEVKFNWYVDGVEV HNAKTKPREE QYNSTYRVVS VLTVLHQDWLNGKEYKCKVS NKALPAPIEK TISKAKGQPR EPQVYTLPPSRDELTKNQVS LTCLVKGFYP SDIAVEWESN GQPENNYKTTPPVLDSDGSF FLYSKLTVDK SRWQQGNVFS CSVMHEALHN HYTQKSLSLS PGK TALL-1 124MFHDCKWDLL TKQWVCHGLG SGSATGGSGS TASSGSGSAT consensusHMFHDCKWDL LTKQWVCHGL GGGGGVDKTH TCPPCPAPEL tandem dimerLGGPSVFLFP PKPKDTLMIS RTPEVTCVVV DVSHEDPEVKFNWYVDGVEV HNAKTKPREE QYNSTYRVVS VLTVLHQDWLNGKEYKCKVS NKALPAPIEK TISKAKGQPR EPQVYTLPPSRDELTKNQVS LTCLVKGFYP SDIAVEWESN GQPENNYKTTPPVLDSDGSF FLYSKLTVDK SRWQQGNVFS CSVMHEALHN HYTQKSLSLS PGK

TABLE 6  Sequences of oligonucleotides used in peptibody construction.Oligo- SEQ nucleotide ID ID number NO Sequence 2517-24 71TAT GCC GGG TAC TTG TTT CCC GTT CCC GTG GGA ATG CACTCA CGC TGG TGG AGG CGG TGG GG 2517-25 72TCG ACC CCA CCG CCT CCT GGA GCG TGA GTG CAT TCC CACGGG AAG CCG AAA CAA GTA CCC GGC A 2517-26 73TAT GTG GGG TGC TTG TTG GCC GTT CCC GTG GGA ATG TTTCAA AGA AGG TGG AGG CGG TGG GG 2517-27 74TCG ACC CCA CCG CCT CCA CCT TCT TTG AAA CAT TCCCACGGG AAC GGC CAA CAAGCA CCC CAC A 2517-28 75TAT GGT TCC GTT CTG TGA CCT GCT GAC TAA ACA CTG TTTCGA AGC TGG TGG AGG CGG TGG GG 2517-29 76TCG ACC CCA CCG CCT CCA CCA GCT TCG AAA CAG TGT TTAGTC AGC AGG TCA CAGAAC GGA ACC A 2517-30 77TAT GGG TTC TCG TTG TAA ATA CAA ATG GGA CGT TCT GACTAA ACA GTG TTT CCA CCA CGG TGG AGG CGG TGG GG 2517-31 78TCG ACC CCA CCG CCT CCA CCG TGG TGG AAA CAC TGT TTAGTC AGA ACG TCC CAT TTG TAT TTA CAA CGA GAA CCC A 2517-32 79TAT GCT GCC GGG TTG TAA ATG GGA CCT GCT GAT CAA ACAGTG GGT TTG TGA CCC GCT GGG TGG AGG CGG TGG GG 2517-33 80TCG ACC CCA CCG CCT CCA CCC AGC GGG TCA CAA ACC CACTGT TTG ATC AGC AGG TCC CAT TTA CAA CCC GGC AGC A 2517-34 81TAT GTC TGC TGA CTG TTA CTT CGA CAT CCT GAC TAA ATCTGA CGT TTG TAC TTC TTC TGG TGG AGG CGG TGG GG 2517-35 82TCG ACC CCA CCG CCT CCA CCA GAA GAA GTA CAA ACG TCAGAT TTA GTC AGG ATG TCG AAG TAA CAG TCA GCA GAC A 2517-36 83TAT GTC TGA CGA CTG TAT GTA CGA CCA GCT GAC TCG TATGTT CAT CTG TTC TAA CCT GGG TGG AGG CGG TGG GG 2517-37 84TCG ACC CCA CCG CCT CCA CCC AGG TTA GAA CAG ATG AACATA CGA GTC AGC TGG TCG TAC ATA CAG TCG TCA GAC A 2521-92 85TAT GGA CCT GAA CTG TAA ATA CGA CGA ACT GAC TTA CAAAGA ATG GTG TCA GTT CAA CGG TGG AGG CGG TGG GG 25221-93  86TCG ACC CCA CCG CCT CCA CCG TTG AAC TGA CAC CAT TCTTTG TAA GTC AGTTCG TCG TAT TTA CAG TTC AGG TCC A 2521-94 87TAT GTT CCA CGA CTG TAA ATA CGA CCT GCT GAC TCG TCAGAT GGT TTG TCA CGG TCT GGG TGG AGG CGG TGG GG 2521-95 88TCG ACC CCA CCG CCT CCA CCC AGA CCG TGA CAA ACC ATCTGA CGA GTC AGC AGG TCG TAT TTA CAG TCG TGG AAC A 2521-96 89TAT GCG TAA CCA CTG TTT CTG GGA CCA CCT GCT GAA ACAGGA CAT CTG TCC GTC TCC GGG TGG AGG CGG TGG GG 2521-97 90TCG ACC CCA CCG CCT CCA CCC GGA GAC GGA CAG ATG TCCTGT TTC AGC AGG TGG TCC CAG AAA CAG TGG TTA CGC A 2521-98 91TAT GGC TAA CCA GTG TTG GTG GGA CTC TCT GCT GAA AAAAAA CGT TTG TGA ATT CTT CGG TGG AGG CGG TGG GG 2521-99 92TCG ACC CCA CCG CCT CCA CCG AAG AAT TCA CAA ACG TTTTTT TTC AGC AGA GAG TCC CAC CAA CAC TGG TTA GCC A 2551-48 93TAT GTT CCA CGA CTG CAA ATG GGA CCT GCT GAC CAA ACAGTG GGT TTG CCA CGG TCT GGG TGG AGG CGG TGG GG 2551-49 94TCG ACC CCA CCG CCT CCA CCC AGA CCG TGG CAA ACC CACTGT TTG GTC AGC AGG TCC CAT TTG CAG TCG TGG AAC ApAMG21-RANK-Fc Vector

pAMG21. The expression plasmid pAMG21 (ATCC accession no. 98113) can bederived from the Amgen expression vector pCFM1656(ATCC#69576) which inturn be derived from the Amgen expression vector system described inU.S. Pat. No. 4,710,473. The pCFM1656 plasmid can be derived from thedescribed pCFM836 plasmid (U.S. Pat. No. 4,710,473) by:

-   -   destroying the two endogenous NdeI restriction sites by end        filling with T4 polymerase enzyme followed by blunt end        ligation;    -   replacing the DNA sequence between the unique AatII and ClaI        restriction sites containing the synthetic P_(L) promoter with a        similar fragment obtained from pCFM636 (U.S. Pat. No. 4,710,473)        containing the P_(L) promoter (see SEQ ID NO: 95 below); and    -   substituting the small DNA sequence between the unique ClaI and        KpnI restriction sites with the oligonucleotide having the        sequence of SEQ ID NO: 96.

SEQ ID NO: 95: AatII 5′CTAATTCCGCTCTCACCTACCAAACAATGCCCCCCTGCAAAAAATAAATTCATAT- 3′TGCAGATTAAGGCGAGAGTGGATGGTTTGTTACGGGGGGACGTMITATTTAAGTATA-   -AAAAAACATACAGATAACCATCTGCGGTGATAAATTATCTCTGGCGGTGTTGACATAAA-   -TTTTTIGTATGTCTATTGGTAGACGCCACTATTTAATAGAGACCGCCACAACTGTATTT-   -TACCACTGGCGGTGATACTGAGCACAT 3′    -ATGGTGACCGCCACTATGACTCGTGTAGC 5′                                ClaI SEQ ID NO: 96: 5′CGATTTGATTCTAGAAGGAGGAATAACATATGGTTAACGCGTTGGAATTCGGTAC 3′ 3′TAAACTAAGATCTTCCTCCTTATTGTATACCAATTGCGCAACCTTAAGC 5′   ClaI                                          KpnI

The expression plasmid pAMG21 can then be derived from pCFM1656 bymaking a series of site-directed base changes by PCR overlappingoligonucleotide mutagenesis and DNA sequence substitutions. Startingwith the BglII site (plasmid by #180) immediately 5′ to the plasmidreplication promoter P _(copB) and proceeding toward the plasmidreplication genes, the base pair changes are as shown in Table 7 below.

TABLE 7  Base pair changes resulting in pAMG21 bp in  bp changedpAMG21 bp # pCFM1656 to in pAMG21 # 204 T/A C/G # 428 A/T G/C # 509 G/CA/T # 617 — insert two G/C bp # 679 G/C T/A # 980 T/A C/G # 994 G/C A/T# 1004 A/T C/G # 1007 C/G T/A # 1028 A/T T/A # 1047 C/G T/A # 1178 G/CT/A # 1466 G/C T/A # 2028 G/C bp deletion # 2187 C/G T/A # 2480 A/T T/A# 2499-2502 AGTG GTCA TCAC CAGT # 2642 TCCGAGC 7 bp deletion AGGCTCG #3435 G/C A/T # 3446 G/C A/T # 3643 A/T T/A

The DNA sequence between the unique AatII (position #4364 in pCFM1656)and SacII (position #4585 in pCFM1656) restriction sites is substitutedwith the DNA sequence below (SEQ ID NO: 97):

[AatII sticky end]               5′     GCGTAACGTATGCATGGTCTCC-(position #4350 in pAMG21)       3′ TGCACGCATTGCATACGTACCAGAGG--CCATGCGAGAGTAGGGAACTGCCAGGCATCAAATAAAACGAAAGGCTCAGTCGAAAGACT--GGTACGCTCTCATCCCTTGACGGTCCGTAGTTTATTTTGCTTTCCGAGTCAGCTTTCTGA--GGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCCTGAGTAGGACAAATCCGC--CCCGGAAAGCAAAATAGACAACAAACAGCCACTTGCGAGAGGACTCATCCTGTTTAGGCG--CGGGAGCGGATTTGAACGTTGCGAAGCAACGGCCCGGAGGGTGGCGGGCAGGACGCCCGC--GCCCTCGCCTAAACTTGCAACGCTTCGTTGCCGGGCCTCCCACCGCCCGTCCTGCGGGCG--CATAAACTGCCAGGCATCAAATTAAGCAGAAGGCCATCCTGACGGATGGCCTTTTTGCGT--GTATTTGACGGTCCGTAGTTTAATTCGTCTTCCGGTAGGACTGCCTACCGGAAAAACGCA-                                                 AatII-TTCTACAAACTCTTTTGTTTATTTTTCTAAATACATTCAAATATGGACGTCGTACTTAAC--AAGATGTTTGAGAAAACAAATAAAAAGATTTATGTAAGTTTATACCTGCAGCATGAATTG--TTTTAAAGTATGGGCAATCAATTGCTCCTGTTAAAATTGCTTTAGAAATACTTTGGCAGC--AAAATTTCATACCCGTTAGTTAACGAGGACAATTTTAACGAAATCTTTATGAAACCGTCG--GGTTTGTTGTATTGAGTTTCATTTGCGCATTGGTTAAATGGAAAGTGACCGTGCGCTTAC--CCAAACAACATAACTCAAAGTAAACGCGTAACCAATTTACCTTTCACTGGCACGCGAATG--TACAGCCTAATATTTTTGAAATATCCCAAGAGCTTTTTCCTTCGCATGCCCACGCTAAAC--ATGTCGGATTATAAAAACTTTATAGGGTTCTCGAAAAAGGAAGCGTACGGGTGCGATTTG--ATTCTTTTTCTCTTTTGGTTAAATCGTTGTTTGATTTATTATTTGCTATATTTATTTTTC--TAAGAAAAAGAGAAAACCAATTTAGCAACAAACTAAATAATAAACGATATAAATAAAAAG--GATAATTATCAACTAGAGAAGGAACAATTAATGGTATGTTCATACACGCATGTAAAAATA--CTATTAATAGTTGATCTCTTCCTTGTTAATTACCATACAAGTATGTGCGTACATTTTTAT--AACTATCTATATAGTTGTCTTTCTCTGAATGTGCAAAACTAAGCATTCCGAAGCCATTAT--TTGATAGATATATCAACAGAAAGAGACTTACACGTTTTGATTCGTAAGGCTTCGGTAATA--TAGCAGTATGAATAGGGAAACTAAACCCAGTGATAAGACCTGATGATTTCGCTTCTTTAA--ATCGTCATACTTATCCCTTTGATTTGGGTCACTATTCTGGACTACTAAAGCGAAGAAATT--TTACATTTGGAGATTTTTTATTTACAGCATTGTTTTCAAATATATTCCAATTAATCGGTG--AATGTAAACCTCTAAAAAATAAATGTCGTAACAAAAGTTTATATAAGGTTAATTAGCCAC--AATGATTGGAGTTAGAATAATCTACTATAGGATCATATTTTATTAAATTAGCGTCATCAT--TTACTAACCTCAATCTTATTAGATGATATCCTAGTATAAAATAATTTAATCGCAGTAGTA--AATATTGCCTCCATTTTTTAGGGTAATTATCCAGAATTGAAATATCAGATTTAACCATAG--TTATAACGGAGGTAAAAAATCCCATTAATAGGTCTTAACTTTATAGTCTAAATTGGTATC--AATGAGGATAAATGATCGCGAGTAAATAATATTCACAATGTACCATTTTAGTCATATCAG--TTACTCCTATTTACTAGCGCTCATTTATTATAAGTGTTACATGGTAAAATCAGTATAGTC--ATAAGCATTGATTAATATCATTATTGCTTCTACAGGCTTTAATTTTATTAATTATTCTGT--TATTCGTAACTAATTATAGTAATAACGAAGATGTCCGAAATTAAAATAATTAATAAGACA--AAGTGTCGTCGGCATTTATGTCTTTCATACCCATCTCTTTATCCTTACCTATTGTTTGTC--TTCACAGCAGCCGTAAATACAGAAAGTATGGGTAGAGAAATAGGAATGGATAACAAACAG--GCAAGTTTTGCGTGTTATATATCATTAAAACGGTAATAGATTGACATTTGATTCTAATAA--CGTTCAAAACGCACAATATATAGTAATTTTGCCATTATCTAACTGTAAACTAAGATTATT--ATTGGATTTTTGTCACACTATTATATCGCTTGAAATACAATTGTTTAACATAAGTACCTG--TAACCTAAAAACAGTGTGATAATATAGCGAACTTTATGTTAACAAATTGTATTCATGGAC--TAGGATCGTACAGGTTTACGCAAGAAAATGGTTTGTTATAGTCGATTAATCGATTTGATT--ATCCTAGCATGTCCAAATGCGTTCTTTTACCAAACAATATCAGCTAATTAGCTAAACTAA--CTAGATTTGTTTTAACTAATTAAAGGAGGAATAACATATGGTTAACGCGTTGGAATTCGA--GATCTAAACAAAATTGATTAATTTCCTCCTTATTGTATACCAATTGCGCAACCTTAAGCT-                                                 SacII-GCTCACTAGTGTCGACCTGCAGGGTACCATGGAAGCTTACTCGAGGATCCGCGGAAAGAA--CGAGTGATCACAGCTGGACGTCCCATGGTACCTTCGAATGAGCTCCTAGGCGCCTTTCTT--GAAGAAGAAGAAGAAAGCCCGAAAGGAAGCTGAGTTGGCTGCTGCCACCGCTGAGCAATA--CTTCTTCTTCTTCTTTCGGGCTTTCCTTCGACTCAACCGACGACGGTGGCGACTCGTTAT--ACTAGCATAACCCCTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTGCTGAAAGGAGG--TGATCGTATTGGGGAACCCCGGAGATTTGCCCAGAACTCCCCAAAAAACGACTTTCCTCC--AACCGCTCTTCACGCTCTTCACGC 3′          [SacII sticky end]-TTGGCGAGAAGTGCGAGAAGTG 5′        (position #5904 in pAMG21)

During the ligation of the sticky ends of this substitution DNAsequence, the outside AatII and SacII sites are destroyed. There areunique AatII and SacII sites in the substituted DNA.

A gene encoding human RANK fused to the N-terminus of Fc was ligatedinto pAMG21 as an NdeI to BamHI fragment to generate Amgen Strain #4125.This construct was modified to insert a valine codon at the junction ofRANK and Fc. The adjacent valine and aspartate codons create a uniqueSalI site. This allows for the fusion of peptides at the N-terminus ofFc3 between the unique NdeI and SalI sites. The RANK sequence is deletedupon insertion of a new NdeI-SalI fragment. The sequence of the vectoris given in FIG. 5A through 5M.

GM221 (Amgen #2596).

The Amgen host strain #2596 is an E. coli K-12 strain derived from Amgenstrain #393, which is a derivative of E. coli W1485, obtained from theE. coli Genetic Stock Center, Yale University, New Haven, Conn. (CGSCstrain 6159). It has been modified to contain both the temperaturesensitive lambda repressor cI857s7 in the early ebg region and thelacI^(Q) repressor in the late ebg region (68 minutes). The presence ofthese two repressor genes allows the use of this host with a variety ofexpression systems, however both of these repressors are irrelevant tothe expression from luxP_(R). The untransformed host has no antibioticresistances.

The ribosome binding site of the cI857s7 gene has been modified toinclude an enhanced RBS. It has been inserted into the ebg operonbetween nucleotide position 1170 and 1411 as numbered in Genbankaccession number M64441Gb_Ba with deletion of the intervening ebgsequence. The sequence of the insert is shown below with lower caseletters representing the ebg sequences flanking the insert shown below(SEQ ID NO: 98):

ttattttcgtGCGGCCGCACCATTATCACCGCCAGAGGTAAACTAGTCAACACGCACGGTGTTAGATATTTATCCCTTGCGGTGATAGATTGAGCACATCGATTTGATTCTAGAAGGAGGGATAATATATGAGCACAAAAAAGAAACCATTAACACAAGAGCAGCTTGAGGACGCACGTCGCCTTAAAGCAATTTATGAAAAAAAGAAAAATGAACTTGGCTTATCCCAGGAATCTGTCGCAGACAAGATGGGGATGGGGCAGTCAGGCGTTGGTGCTTTATTTAATGGCATCAATGCATTAAATGCTTATAACGCCGCATTGCTTACAAAAATTCTCAAAGTTAGCGTTGAAGAATTTAGCCCTTCAATCGCCAGAGAATCTACGAGATGTATGAAGCGGTTAGTATGCAGCCGTCACTTAGAAGTGAGTATGAGTACCCTGTTTTTTCTCATGTTCAGGCAGGGATGTTCTCACCTAAGCTTAGAACCTTTACCAAAGGTGATGCGGAGAGATGGGTAAGCACAACCAAAAAAGCCAGTGATTCTGCATTCTGGCTTGAGGTTGAAGGTAATTCCATGACCGCACCAACAGGCTCCAAGCCAAGCTTTCCTGACGGAATGTTAATTCTCGTTGACCCTGAGCAGGCTGTTGAGCCAGGTGATTTCTGCATAGCCAGACTTGGGGGTGATGAGTTTACCTTCAAGAAACTGATCAGGGATAGCGGTCAGGTGTTTTTACAACCACTAAACCCACAGTACCCAATGATCCCATGCAATGAGAGTTGTTCCGTTGTGGGGAAAGTTATCGCTAGTCAGTGGCCTGAAGAGACGTTTGGCTGATAGACTAGTGGATCCACTAGTgtttctgccc

The construct was delivered to the chromosome using a recombinant phagecalled MMebg-cI857s7enhanced RBS #4 into F′tet/393. After recombinationand resolution only the chromosomal insert described above remains inthe cell. It was renamed F′tet/GM101. F′tet/GM101 was then modified bythe delivery of a lacI^(Q) construct into the ebg operon betweennucleotide position 2493 and 2937 as numbered in the Genbank accessionnumber M64441Gb_Ba with the deletion of the intervening ebg sequence.The sequence of the insert is shown below with the lower case lettersrepresenting the ebg sequences flanking the insert (SEQ ID NO: 99) shownbelow:

ggcggaaaccGACGTCCATCGAATGGTGCAAAACCTTTCGCGGTATGGCATGATAGCGCCCGGAAGAGAGTCAATTCAGGGTGGTGAATGTGAAACCAGTAACGTTATACGATGTCGCAGAGTATGCCGGTGTCTCTTATCAGACCGTTTCCCGCGTGGTGAACCAGGCCAGCCACGTTTCTGCGAAAACGCGGGAAAAAGTCGAAGCGGCGATGGCGGAGCTGAATTACATTCCCAACCGCGTGGCACAACAACTGGCGGGCAAACAGTCGCTCCTGATTGGCGTTGCCACCTCCAGTCTGGCCCTGCACGCGCCGTCGCAAATTGTCGCGGCGATTAAATCTCGCGCCGATCAACTGGGTGCCAGCGTGGTGGTGTCGATGGTAGAACGAAGCGGCGTCGAAGCCTGTAAAGCGGCGGTGCACAATCTTCTCGCGCAACGCGTCAGTGGGCTGATCATTAACTATCCGCTGGATGACCAGGATGCCATTGCTGTGGAAGCTGCCTGCACTAATGTTCCGGCGTTATTTCTTGATGTCTCTGACCAGACACCCATCAACAGTATTATTTTCTCCCATGAAGACGGTACGCGACTGGGCGTGGAGCATCTGGTCGCATTGGGTCACCAGCAAATCGCGCTGTTAGCGGGCCCATTAAGTTCTGTCTCGGCGCGTCTGCGTCTGGCTGGCTGGCATAAATATCTCACTCGCAATCAAATTCAGCCGATAGCGGAACGGGAAGGCGACTGGAGTGCCATGTCCGGTTTTCAACAAACCATGCAAATGCTGAATGAGGGCATCGTTCCCACTGCGATGCTGGTTGCCAACGATCAGATGGCGCTGGGCGCAATGCGCGCCATTACCGAGTCCGGGCTGCGCGTTGGTGCGGATATCTCGGTAGTGGGATACGACGATACCGAAGACAGCTCATGTTATATCCCGCCGTTAACCACCATCAAACAGGATTTTCGCCTGCTGGGGCAAACCAGCGTGGACCGCTTGCTGCAACTCTCTCAGGGCCAGGCGGTGAAGGGCAATCAGCTGTTGCCCGTCTCACTGGTGAAAAGAAAAACCACCCTGGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATGCAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGACAGTAAGGTACCATAGGATCCaggcacagga

The construct was delivered to the chromosome using a recombinant phagecalled AGebg-LacIQ#5 into F′tet/GM101. After recombination andresolution only the chromosomal insert described above remains in thecell. It was renamed F′tet/GM221. The F′tet episome was cured from thestrain using acridine orange at a concentration of 25 μg/ml in LB. Thecured strain was identified as tetracyline sensitive and was stored asGM221.

Expression in E. coli.

Cultures of each of the pAMG21-Fc-fusion constructs in E. coli GM221were grown at 37° C. in Luria Broth medium. Induction of gene productexpression from the luxPR promoter was achieved following the additionof the synthetic autoinducer N-(3-oxohexanoyl)-DL-homoserine lactone tothe culture media to a final concentration of 20 ng/ml. Cultures wereincubated at 37° C. for a further 3 hours. After 3 hours, the bacterialcultures were examined by microscopy for the presence of inclusionbodies and were then collected by centrifugation. Refractile inclusionbodies were observed in induced cultures indicating that the Fc-fusionswere most likely produced in the insoluble fraction in E. coli. Cellpellets were lysed directly by resuspension in Laemmli sample buffercontaining 10% β-mercaptoethanol and were analyzed by SDS-PAGE. In eachcase, an intense Coomassie-stained band of the appropriate molecularweight was observed on an SDS-PAGE gel.

Example 3 TALL-1 Peptibody Inhibits TALL-1 Mediated B Cell Proliferation

Mouse B lymphocytes were isolated from C57BL/6 spleens by negativeselection. (MACS CD43 (Ly-48) Microbeads, Miltenyi Biotech, Auburn,Calif.). Purified (10⁵) B cells were cultured in MEM, 10% heatinactivated FCS, 5×10⁻⁵M 2-mercaptoethanol, 100 U/ml penicillin, 100μg/ml streptomycin) in triplicate in 96-well flat bottom tissue cultureplates with 10 μg/ml TALL-1 protein and 2 μg/ml of Goat F(ab′)₂anti-mouse IgM (Jackson ImmunoResearch Laboratory, West Grove, Pa.) withthe indicated amount of recombinant TALL-1 peptibody for a period of 4days at 37° C., 5% CO₂. Proliferation was measured by the uptake ofradioactive ³[H] thymidine after an 18-hour incubation period.

Example 4 TALL-1 Peptibody Blocks TALL-1 Binding to its Receptors

Reacti-Gel 6× (Pierce) were pre-coated with human AGP3 (also known asTALL-1, Khare et al., Proc. Natl. Acad. Sci. 97:3370-3375, 2000) andblocked with BSA. 100 pM and 40 pM of AGP3 peptibody samples wereincubated with indicated various concentrations of human AGP3 at roomtemperature for 8 hours before run through the human AGP3-coated beads.The amount of the bead-bound peptibody was quantified by fluorescent(Cy5) labeled goat anti-human-Fc antibody (Jackson Immuno Research). Thebinding signal is proportional to the concentration of free peptibody atbinding equilibrium. Dissociation equilibrium constant (K_(D)) wasobtained from nonlinear regression of the competition curves using adual-curve one-site homogeneous binding model (KinEx™ software). K_(D)is about 4 pM for AGP3 peptibody (SEQ ID NO: 123) binding with humanAGP3 (FIG. 9).

To determine if this AGP3 peptibody can neutralize murine AGP3 bindingas well as human AGP3, a BlAcore neutralizing assay was utilized. Allexperiments were performed on a BlAcore 3000 at room temperature. HumanTACI-Fc protein (Xia et al, J. Exp. Med. 192, 137-144, 2000) wasimmobilized to a Bl chip using 10 mM Acetate pH 4.0 to a level of2900RU. A blank flow cell was used as a background control. Using arunning buffer of PBS (without calcium or magnesium) containing 0.005%P20, 1 nM recombinant human AGP3 (in running buffer plus, 0.1 mg/ml BSA)was incubated without and with indicated various amount of AGP3peptibody (x axis) before injected over the surface of the receptor.Regeneration was performed using 8 mM glycine pH 1.5 for 1 minute, 25 mM3-[cyclohexylamino]-1-propanesulfonic acid (CAPS) pH 10.5, 1 M NaCl for1 minute. For determination of murine AGP3 binding, human his-taggedTACI was immobilized to 1000 RU in the above buffer. 5 nM recombinantmurine AGP3 (in running buffer plus, 0.1 mg/ml BSA) was incubatedwithout and with the various amounts indicated in FIGS. 11A and B ofAGP3 peptibody (x axis) before injected over the surface of thereceptor. Regeneration was performed with 10 mM HCl pH2, twice for 30seconds. Relative binding of both human and murine AGP3 at presence vsabsence of AGP3 peptibody (SEQ ID NO: 123) was measured (y axis).Relative binding response was determined as (RU-RU blank/RUo-RU blank).The AGP3 peptibody (SEQ ID NO: 123) inhibited both human and murine AGP3binding to its receptor TACI (FIGS. 10A and 10B).

To examine if this AGP3 peptibody blocks AGP3 binding to all threereceptors (TACI, BCMA and BAFFR), recombinant soluble receptor TACI,BCMA and BAFFR proteins were immobilized to CM5 chip. Using 10 mMacetate, pH4, human TACI-Fe was immobilized to 6300 RU, human BCMA-Fc to5000 RU, and BAFFR-Fc to 6000 RU. 1 nM of recombinant human AGP3 (inrunning buffer containing 0.1 mg/ml BSA and 0.1 mg/ml Heparin) or 1 nMrecombinant APRIL protein (Yu, et al., Nat. Immunol., 1:252-256, 2000)were incubated with indicated amount of AGP3 peptibody before injectionover each receptor surface. Regeneration for the AGP3 experiment wasdone with 8 mM glycine, pH 1.5, for 1 minute, followed by 25 mM CAPS, pH10.5, 1M NaCl for 1 minute. Regeneration for the APRIL experiment wasperformed with 8 mM glycine, pH 2, for one minute, followed by 25 mMCAPS, pH 10.5, 1 M NaCl for one minute. Relative binding of AGP3 orAPRIL was measured. AGP3 peptibody (SEQ ID NO: 123) blocked AGP3 bindingto all three receptors (FIG. 11A). AGP3 peptibody didn't affect APRILbinding to the receptors (FIG. 11B).

Example 5 AGP3 Peptibody Blocks AGP3 Mediated B Cell Proliferation

Mouse B lymphocytes were isolated from C57BL/6 spleens by negativeselection. (MACS CD43 (Ly-48) Microbeads, Miltenyi Biotech, Auburn,Calif.). Purified (10⁵) B cells were cultured in minimal essentialmedium (MEM), 10% heat inactivated fetal calf serum (FCS), 5×10⁻⁵M2-mercaptoethanol, 100 U/ml penicillin, 100 μg/ml streptomycin) intriplicate in 96-well flat bottom tissue culture plates with 10 ng/mlAGP3 (TALL-1) protein and 2 μg/ml of Goat F(ab′)₂ anti-mouse IgM(Jackson ImmunoResearch Laboratory, West Grove, Pa.) with the indicatedamount of recombinant AGP3 peptibody (SEQ ID NO: 123) for a period of 4days at 37° C., 5% CO₂. Proliferation was measured by the uptake ofradioactive ³[H] thymidine after an 18-hour incubation period.

Example 6 AGP3 Peptibody on AGP3-Stimulated Ig Production in Mice

Mice (Balb/c females of 9-14 weeks of age and 19-21 g of weight) werepurchased from Charles River Laboratories, Wilmington, Mass. Mice (n=10)were treated i.p. with 1 mg/Kg of human AGP3 once a day for fiveconsecutive days followed by 5 mg/Kg or 0.5 mg/Kg of AGP3 peptibody (SEQID NO: 123) or by saline or by 5 mg/Kg of human Fc. Other mice were leftuntreated. Mice were sacrificed on the sixth day to measure serum IgMand IgA, which were measured by ELISA. Briefly, plates were coated withcapture antibodies specific for IgM or IgA (Southern BiotechnologyAssociates, Birmingham, Ala.), blocked, and added with dilutions ofstandard (IgM from Calbiochem, San Diego, Calif. and IgA from SouthernBiotechnology Associates) or test samples. Captured Ig were revealedusing biotinylated antibodies specific for IgM or IgA (SouthernBiotechnology Associates), neutravidin-conjugated peroxidase (Pierce,Rockford, Ill.), and tetramethylbenzidine (TMB) microwell peroxidasesubstrate (KPL, Gaithersburg, Md.). Optical densities were quantitatedin a Thermomax ELISA reader (Molecular Devices, Menlo Park, Calif.).

Human AGP3-stimulated increase in serum levels of IgM and IgA wasblocked by 5 mg/Kg of the anti-AGP3 peptibody (SEQ ID NO: 123) and notby 0.5 mg/Kg (FIGS. 12A and 12B).

Example 7 AGP3 Peptibody Reduced Spleen B Cell Number in Mice

Mice (as above, n=7) were treated i.p. for seven consecutive days with 5mg/Kg or 1.5 mg/Kg or 0.5 mg/Kg of AGP3 peptibody (SEQ ID NO: 123) orwith saline or with 5 mg/Kg of human Fc. Mice were sacrificed on theeighth day to count spleen B cell number. Spleens were collected insaline and gently disrupted by manual homogenization to yield a cellsuspension. The total cell number was obtained with a HE counter(Technicon, Tarrytown, N.Y.). Percentages of B cells were derived byimmunofluorescence double staining and flow cytometry using fluoresceinisothiocyanate (FITC)-conjugated and phycoerythrin (PE)-conjugated Abagainst CD3 and B220, respectively (PharMingen, San Diego, Calif.) and aFACScan analyser (Becton and Dickinson, Mountain View, Calif.). B cellswere identified for being CD3-B220+. At all doses, the AGP3 peptibody(SEQ ID NO: 123) decreased spleen B cell number in a dose-responsefashion (FIGS. 12A and 12B) (SEQ ID NO: 123).

TABLE 8 AGP3 Pb Reduces B Cell Number in Normal Mice dose spleen B celln = 7 (1/day × 7) (1 × 10e6) SD t test saline 51.3 9.6 Fc   5 mg/Kg 45.57.1 Peptibody   5 mg/Kg 20.1 3.8 1.37856E−05 1.5 mg/Kg 22.6 6.95.10194E−05 0.5 mg/Kg 25.8 3.6 0.000111409

Example 8 AGP3 Peptibody Reduced Arthritis Severity in Mouse CIA Model

Eight to 12 week old DBA/1 mice (obtained from Jackson Laboratories, BarHarbor, Me.) were immunized with bovine collagen type II (bCII)(purchased from University of Utah), emulsified in complete Freundsadjuvant (Difco) intradermally at the base of tail. Each injection was100 μl containing 100 μg of bCII. Mice were boosted 3 weeks after theinitial immunization with bCII emulsified in incomplete Freundsadjuvant. Treatment was begun from the day of booster immunization for 4weeks. Mice were examined for the development of arthritis. As describedbefore (Khare et al., J. Immunol, 155: 3653-9, 1995), all four paws wereindividually scored from 0-3. Therefore arthritis severity could varyfrom 0 to 12 for each animal. AGP3 (SEQ ID NO: 123) peptibody treatmentsignificantly reduced the severity of arthritic scores (FIG. 13).

Serum samples were taken one week after final treatment (day 35) for theanalysis of anti-collagen antibody level. High binding ELISA plates(Immulon, Nunc) were coated with 50 μl of 4 μg/ml solution of bovine CIIin carbonate buffer and plated were kept in cold overnight in therefrigerator. Plates were washed three times with cold water. 75 μl ofblocking solution made up of PBS/0.05% tween 20/1% BSA was used to blocknon-specific binding for an hour. Samples were diluted (in blockingbuffer) in dilution plates at 1:25, 1:100, 1:400, and 1:1600 and 25 μlof these samples were added to each well of the ELISA plate for a finaldilution of 100, 400, 1600, and 6400 with a final volume of 100 μl/well.After incubation at room temperature for 3 hours, plates were washedthree times again. 100 μl of secondary antibody diluted in blockingbuffer (rat anti-mouse IgM, IgG2a, IgG2b, IgG1, IgG3-HRP) was added toeach well and plates were incubated for at least 2 hours. Plates werewashed four times. 100 μl of TMB solution (Sigma) was added to each welland the reaction was stopped using 50 μl of 25% sulfuric acid. Plateswere read using an ELISA plate reader at 450 nm. OD was compared with astandard pool representing units/ml. AGP3 peptibody (SEQ ID NO: 123)treatment reduced serum anti-collagen II IgG1, IgG3, IgG2a, and IgG2blevels compared to PBS or Fc control treatment groups (FIG. 14).

Example 9 Treatment of AGP3 Peptibody in NZB/NZW Lupus Mice

Five month old lupus prone NZBx NZBWF1 mice were treated i.p. 3×/weekfor 8 weeks with PBS or indicated doses of AGP3 peptibody or human Fcproteins. Prior to the treatment, animals were pre-screened for proteinin the urine with Albustix reagents strips (Bayer AG). Mice havinggreater than 100 mg/dl of protein in the urine were not included in thestudy. Protein in the urine was evaluated monthly throughout the life ofthe experiment. AGP3 peptibody (SEQ ID NO: 123) treatment led to delayof proteinuria onset and improved survival (FIGS. 15A and 15B).

AGP3 peptibody treatment reduced B cell number in mice. Balb/c micereceived 7 daily intraperitoneal injections of indicated amount of AGP3peptibody (SEQ ID NO: 123) or human Fc protein. On day 8, spleens werecollected, and subject to FACS analysis for B220+B cells as set for inTable 8.

TABLE 8 AGP3 Pb Reduces B Cell Number in Normal Mice dose Spleen B celln = 7 (1/day × 7) (1 × 10e6) SD t test saline 51.3 9.6 Fc   5 mg/Kg 45.57.1 Peptibody   5 mg/Kg 20.1 3.8 1.37856E−05 1.5 mg/Kg 22.6 6.95.10194E−05 0.5 mg/Kg 25.8 3.6 0.000111409

The invention now being fully described, it will be apparent to one ofordinary skill in the art that many changes and modifications can bemade thereto, without departing from the spirit and scope of theinvention as set forth herein.

What is claimed is:
 1. A method of treating systemic lupus erythematosusin a subject in need thereof comprising administering to the subject aTALL-1 binding molecule comprising the amino acid sequencef¹f²f³Kf₅Df⁷Lf⁹f¹⁰Qf¹²f¹³f¹⁴ (SEQ. ID. NO: 109), wherein: f¹, f², and f³are absent or are amino acid residues; f⁵ is W; f⁷ is an amino acidresidue; f⁹ is T or I; f¹⁰ is K, R, or H; f¹² is C, a neutralhydrophobic residue, or a basic residue; f¹³ is C, a neutral hydrophobicresidue or is absent; and f¹⁴ is any amino acid residue or is absent;provided that only one of f¹, f², and f³ may be C, and only one of f¹²,f¹³, and f¹⁴ may be C.
 2. The method of claim 1, wherein the TALL-1binding molecule comprises the amino acid sequencef¹f²f³KWDf⁷Lf⁹KQf¹²f¹³f¹⁴ (SEQ ID NO: 125).
 3. The method of claim 2,wherein the TALL-1 binding molecule comprises the amino acid sequenceLPGCKWDLLIKQWVCDPL (SEQ ID NO:33).
 4. The method of claim 3, wherein theTALL-1 binding molecule comprises the amino acid sequence of SEQ IDNO:44.
 5. The method of claim 4, wherein the TALL-1 binding moleculecomprises the amino acid sequence of SEQ ID NO:12.
 6. A method oftreating systemic lupus erythematosus in a subject in need thereofcomprising administering to the subject a TALL-1 binding moleculecomprising(X¹)_(a)—V¹—(X²)_(b) and multimers thereof, wherein: V¹ is an Fc domain;X¹ and X² are each independently selected from -(L¹)_(c)-P¹-(L²)_(d)-P²,-(L¹)_(c)-P¹-(L²)_(d)-P²-(L³)_(e)-P³,-(L¹)_(c)-P¹-(L²)_(d)-P²-(L³)_(e)-P³-(L⁴)_(f)-P⁴; one or more of P¹, P²,P³, and P⁴ each independently comprise a TALL-1 modulating domaincomprising the amino acid sequencef¹-f²-f³-Lys-f⁵-Asp-f⁷-Leu-f⁹-f¹⁰-Gln-f¹²-f¹³-f¹⁴ (SEQ ID NO: 109), andhaving a maximum length of 40 amino acids; f¹ and f² are absent or areamino acid residues; f³ is Cys; f⁵ is Trp, Tyr, or Phe; f⁷ is an aminoacid residue; f⁹ is Thr or Ile; f¹⁰ is Lys, Arg, or His; f¹² is aneutral hydrophobic residue, or a basic residue; f¹³ is Val; and f¹⁴ isCys; L¹, L², L³, and L⁴ are each independently linkers, wherein eachlinker is selected from a peptide linker, alkyl linker, or a derivativethereof; and a, b, c, d, e, and f are each independently 0 or 1,provided that at least one of a and b is
 1. 7. The method of claim 6,wherein: f⁵ is Trp; f⁷ is Leu; and f¹⁰ is Lys.
 8. The method of claim 6,wherein one or more of P¹, P², P³, and P⁴ each independently comprises(SEQ ID NO: 125) f¹-f²-f³-Lys-Trp-Asp-f⁷-Leu-f⁹-Lys-Gln-f¹²-f⁴³-f¹⁴.


9. A method of treating systemic lupus erythematosusin a subject in needthereof comprising administering to the subject a TALL-1 bindingmolecule comprising the formula:P²-(L²)-P¹-(L¹)-V¹ wherein: P¹ comprises the amino acid sequence of SEQID NO:33; P² comprises the amino acid sequence of SEQ ID NO:125; and L¹and L² are peptide linkers; and V¹ is a vehicle.
 10. The method of claim9, wherein P¹-(L¹)-V¹ comprises the amino acid sequence of SEQ ID NO:44.11. The method of 9, wherein L¹ comprises a Gly₅ peptide linker.
 12. Themethod of claim 9, wherein P¹-(L¹) comprises the amino acid sequence ofSEQ ID NO:12.
 13. The method of claim 9, wherein V¹ is an Fc domain. 14.The method of claim 9, wherein P¹-(L¹)-V¹ comprises the amino acidsequence of SEQ ID NO:115.
 15. The method of claim 9, wherein L²comprises an amino acid sequence selected from the group consisting ofSEQ ID NO:59 and SEQ ID NO:193.